scholarly journals Role of Plant Cell Conditioned Medium in the Phenotypic Expression of Nitrogenase Activity of Rhizobium trifolii Strain T1

1980 ◽  
Vol 33 (5) ◽  
pp. 613 ◽  
Author(s):  
Minocher Reporter ◽  
Mary L Skotnicki ◽  
Barry G Rolfe
Keyword(s):  
Oxidative Phosphorylation ◽  
Conditioned Medium ◽  
Plant Cell ◽  
Nitrogenase Activity ◽  
Phenotypic Expression ◽  
Atp Synthesis ◽  
Atp Production ◽  
Cell Conditioned Medium

The influence of substances from a conditioned medium of cultured plant cells on nitrogenase activity, respiration and ATP synthesis was investigated in R. tri/olii strain Tl. Nitrogenase activity in strain Tl was dependent on the addition of the plant cell conditioned medium. Studies showed that the initial effects of the plant substances on rhizobial cells was to increase their respiration rate and ATP production. Mutants of strain Tl which were uncoupled in their oxidative phosphorylation, were also tested. However, the plant factors had no effect on respiration and ATP synthesis and also failed to elicit in vitro nitrogenase activity in these mutants. It is proposed that these plant factors act by increasing the efficiency of oxidative phosphorylation, making more ATP available, and thus stimulating nitrogenase activity of R. tri/olii cells.

scholarly journals Bisphenol A Altersβ-hCG and MIF Release by Human Placenta: AnIn VitroStudy to Understand the Role of Endometrial Cells

2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
C. Mannelli ◽  
F. Ietta ◽  
C. Carotenuto ◽  
R. Romagnoli ◽  
A. Z. Szostek ◽  
...  
Keyword(s):  
Bisphenol A ◽  
Conditioned Medium ◽  
Human Placenta ◽  
Environmental Chemicals ◽  
Endometrial Cells ◽  
Direct Exposure ◽  
And Control ◽  
Cell Conditioned Medium

A proper fetomaternal immune-endocrine cross-talk in pregnancy is fundamental for reproductive success. This might be unbalanced by exposure to environmental chemicals, such as bisphenol A (BPA). As fetoplacental contamination with BPA originates from the maternal compartment, this study investigated the role of the endometrium in BPA effects on the placenta. To this end,in vitrodecidualized stromal cells were exposed to BPA 1 nM, and their conditioned medium (diluted 1 : 2) was used on chorionic villous explants from human placenta. Parallel cultures of placental explants were directly exposed to 0.5 nM BPA while, control cultures were exposed to the vehicle (EtOH 0.1%). After 24–48 h, culture medium from BPA-treated and control cultures was assayed for concentration of hormone human Chorionic Gonadotropin (β-hCG) and cytokine Macrophage Migration Inhibitory Factor (MIF). The results showed that direct exposure to BPA stimulated the release of both MIF andβ-hCG. These effects were abolished/diminished in placental cultures exposed to endometrial cell-conditioned medium. GM-MS analysis revealed that endometrial cells retain BPA, thus reducing the availability of this chemical for the placenta. The data obtained highlight the importance ofin vitromodels including the maternal component in reproducing the effects of environmental chemicals on human fetus/placenta.

Abstract MP247: Mitochondrial Complex V Is Regulated By GRK2 In The Heart

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Kimberly Ferrero ◽  
Jessica M Pfleger ◽  
Kurt Chuprun ◽  
Eric Barr ◽  
Erhe Gao ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Cardiac Injury ◽  
Atp Synthesis ◽  
Cardiac Metabolism ◽  
Neonatal Rat ◽  
Interacting Proteins ◽  
Atp Production

The GPCR kinase GRK2 is highly expressed the heart; importantly, during cardiac injury or heart failure (HF) both levels and activity of GRK2 increase. The role of GRK2 during HF is canonically studied upstream of β-adrenergic desensitization. However, GRK2 has a large interactome and noncanonical functions for this kinase are being uncovered. We have discovered that in the heart, GRK2 translocates to mitochondria ( mtGRK2 ) following injury and is associated with negative effects on cardiac metabolism. Thus, we have sought to identify the mechanism(s) by which GRK2 can regulate mitochondrial function. We hypothesize that mtGRK2 interacts with proteins which regulate bioenergetics and substrate utilization, and this never-before-described role may partially explain the altered mitochondrial phenotype seen following cardiac injury or HF. Stress-induced mitochondrial translocation of GRK2 was validated in neonatal rat ventricular myocytes, murine heart tissue and a cardiac-derived cell line. Consequently, the GRK2 interactome was mapped basally and under stress conditions in vitro, in vivo , and with tagged recombinant peptides. GRK2-interacting proteins were isolated via immunoprecipitation and analyzed via liquid chromatography-mass spectroscopy (LCMS). Proteomics analysis (IPA; Qiagen) identified mtGRK2 interacting proteins which were also involved in mitochondrial dysfunction. Excitingly, Complexes I, II, IV and V (ATP synthase) of the electron transport chain (ETC) were identified in the subset of mtGRK2-dysfunction partners. Several mtGRK2-ETC interactions were increased following stress, particularly those in Complex V. We further established that mtGRK2 phosphorylates some of the subunits of Complex V, particularly the ATP synthase barrel which is critical for ATP production in the heart. Specific amino acid residues on these subunits have been identified using PTM-LCMS and are currently being validated in a murine model of myocardial infarction. To support these data, we have also determined that alterations in either the levels or kinase activity of GRK2 appear to alter the enzymatic activity of Complex V in vitro , thus altering ATP production. In summary, the phosphorylation of the ATP synthesis machinery by mtGRK2 may be regulating some of the phenotypic effects of injured or failing hearts such as increased ROS production and reduced fatty acid metabolism. Research is ongoing in our lab to elucidate the novel role of GRK2 in regulating mitochondrial bioenergetics and cell death, thus uncovering an exciting, druggable novel target for rescuing cardiac function in patients with injured and/or failing hearts.

scholarly journals Insights into Cadmium-Induced Carcinogenesis through an In Vitro Study Using C3H10T1/2Cl8 Cells: The Multifaceted Role of Mitochondria

2021 ◽  
Vol 22 (19) ◽  
pp. 10837
Author(s):  
Monica Oldani ◽  
Anna Maria Villa ◽  
Marta Manzoni ◽  
Pasquale Melchioretto ◽  
Paolo Parenti ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Oxidative Phosphorylation ◽  
Cell Transformation ◽  
In Vitro Study ◽  
Atp Synthesis ◽  
Cadmium Treatment ◽  
Mouse Embryo Fibroblasts

In this paper, we report the metabolic characterization of two foci, F1 and F3, obtained at the end of Cell Transformation Assay (CTA), performed by treating C3H10T1/2Cl8 mouse embryo fibroblasts with 1 μM CdCl2 for 24 h. The elucidation of the cadmium action mechanism can be useful both to improve the in vitro CTA and to yield insights into carcinogenesis. The metabolism of the two foci was investigated through Seahorse and enzyme activity assays; mitochondria were studied in confocal microscopy and reactive oxygen species were detected by flow cytometry. The results showed that F1 focus has higher glycolytic and TCA fluxes compared to F3 focus, and a more negative mitochondrial membrane potential, so that most ATP synthesis is performed through oxidative phosphorylation. Confocal microscopy showed mitochondria crowded in the perinuclear region. On the other hand, F3 focus showed lower metabolic rates, with ATP mainly produced by glycolysis and damaged mitochondria. Overall, our results showed that cadmium treatment induced lasting metabolic alterations in both foci. Triggered by the loss of the Pasteur effect in F1 focus and by mitochondrial impairment in F3 focus, these alterations lead to a loss of coordination among glycolysis, TCA and oxidative phosphorylation, which leads to malignant transformation.

scholarly journals Insights into cadmium-induced carcinogenesis through an in-vitro study using C3H10T1/2Cl8 cells: the multifaceted role of mitochondria

2021 ◽  
Author(s):  
Monica Oldani ◽  
Anna Maria Villa ◽  
Marta Manzoni ◽  
Pasquale Melchioretto ◽  
Paolo Parenti ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Oxidative Phosphorylation ◽  
Cell Transformation ◽  
Expression Patterns ◽  
In Vitro Study ◽  
Atp Synthesis ◽  
Cadmium Treatment

Abstract In this paper we report the metabolic characterization of two foci, F1 and F3, obtained at the end of Cell Transformation Assay (CTA), performed by treating C3H10T1/2Cl8 mouse embryo fibroblasts with 1 µM CdCl2 for 24 h. The elucidation of cadmium action mechanism can be useful both to improve the in vitro CTA and to yield insights into carcinogenesis. We previously showed that, despite being both completely transformed type III foci, F1 and F3 foci display different morphologies, proliferative behaviors and gene expression patterns. In this work, the metabolism of the two foci was investigated through Seahorse and enzyme activity assays; moreover, mitochondria were studied in confocal microscopy and reactive oxygen species were detected by flow cytometry. Results showed that F1 focus has higher glycolytic and TCA fluxes compared to F3 focus, and a more negative mitochondrial membrane potential (Δψ), so that most ATP synthesis is performed through oxidative phosphorylation. Confocal microscopy showed mitochondria crowded in the perinuclear region. On the other hand, F3 focus showed lower metabolic rates, with ATP mainly produced by glycolysis and damaged mitochondria. On the whole, our results showed that cadmium treatment induced lasting metabolic alterations in both foci. Triggered by the loss of Pasteur effect in F1 focus and by mitochondrial impairment in F3 focus, these alterations lead to a loss of coordination among glycolysis, TCA and oxidative phosphorylation, which leads to malignant transformation.

Ca2+ activation of heart mitochondrial oxidative phosphorylation: role of the F0/F1-ATPase

2000 ◽  
Vol 278 (2) ◽  
pp. C423-C435 ◽  
Author(s):  
Paul R. Territo ◽  
Vamsi K. Mootha ◽  
Stephanie A. French ◽  
Robert S. Balaban
Keyword(s):  
Oxidative Phosphorylation ◽  
Atp Synthesis ◽  
High Energy ◽  
Ruthenium Red ◽  
Maximal Effect ◽  
Production Rates ◽  
Atp Production ◽  
The Matrix

Ca2+ has been postulated as a cytosolic second messenger in the regulation of cardiac oxidative phosphorylation. This hypothesis draws support from the well-known effects of Ca2+ on muscle activity, which is stimulated in parallel with the Ca2+-sensitive dehydrogenases (CaDH). The effects of Ca2+ on oxidative phosphorylation were further investigated in isolated porcine heart mitochondria at the level of metabolic driving force (NADH or Δψ) and ATP production rates (flow). The resulting force-flow (F-F) relationships permitted the analysis of Ca2+ effects on several putative control points within oxidative phosphorylation, simultaneously. The F-F relationships resulting from additions of carbon substrates alone provided a model of pure CaDH activation. Comparing this curve with variable Ca2+ concentration ([Ca2+]) effects revealed an approximate twofold higher ATP production rate than could be explained by a simple increase in NADH or Δψ via CaDH activation. The half-maximal effect of Ca2+ at state 3 was 157 nM and was completely inhibited by ruthenium red (1 μM), indicating matrix dependence of the Ca2+ effect. Arsenate was used as a probe to differentiate between F0/F1-ATPase and adenylate translocase activity by a futile recycling of ADP-arsenate within the matrix, catalyzed by the F0/F1-ATPase. Ca2+increased the ADP arsenylation rate more than twofold, suggesting a direct effect on the F0/F1-ATPase. These results suggest that Ca2+ activates cardiac aerobic respiration at the level of both the CaDH and F0/F1-ATPase. This type of parallel control of both intermediary metabolism and ATP synthesis may provide a mechanism of altering ATP production rates with minimal changes in the high-energy intermediates as observed in vivo.

scholarly journals Expression of the macrophage-specific antigen F4/80 during differentiation of mouse bone marrow cells in culture.

1981 ◽  
Vol 154 (3) ◽  
pp. 713-725 ◽  
Author(s):  
S Hirsch ◽  
J M Austyn ◽  
S Gordon
Keyword(s):  
Conditioned Medium ◽  
Cell Sorting ◽  
Bone Marrow Cells ◽  
Specific Antigen ◽  
Mouse Bone Marrow ◽  
L Cell ◽  
Maturation In Vitro ◽  
Cell Conditioned Medium

We have defined the expression of the macrophages (m phi)-specific antigen (Ag) F4/80 during differentiation in culture. The progenitor cells-the colony-forming unit in culture and cluster-forming cell-lacked Ag F4/80 but gave rise to colonies of F4/80-positive adherent m phi, as shown by fluorescence-activated cell sorting and clonal assays with L cell-conditioned medium as the source of growth factor. Ag F4/80 first appeared on a nonadherent precursor found in mass liquid BM cultures after 3 d. Once adherent, m phi expressed high levels of Ag F4/80 and other markers. The role of L cell-conditioned medium and of adherence on expression of Ag F4/80 was also examined. Clonal analysis of F4/80 and other Ag, Mac-1, and 2.4G2 (FcR) showed that all cells in all independent colonies come to express these markers. These studies establish that F4/80 is a marker for the more mature stages of m phi development and that Ag expression increases progressively during maturation in vitro. Heterogeneity of Ag expression can be ascribed to variation in development and not to independent subsets of the m phi.

Role of calcium in metabolic signaling between cardiac sarcoplasmic reticulum and mitochondria in vitro

2003 ◽  
Vol 284 (2) ◽  
pp. C285-C293 ◽  
Author(s):  
Robert S. Balaban ◽  
Salil Bose ◽  
Stephanie A. French ◽  
Paul R. Territo
Keyword(s):  
Energy Metabolism ◽  
Sarcoplasmic Reticulum ◽  
Atp Synthesis ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Atp Production ◽  
Wide Range ◽  
Reconstituted System

The role of Ca2+ as a cytosolic signaling molecule between porcine cardiac sarcoplasmic reticulum (SR) ATPase and mitochondrial ATP production was evaluated in vitro. The Ca2+ sensitivity of these processes was determined individually and in a reconstituted system with SR and mitochondria in a 0.5:1 protein-to-cytochrome aa 3 ratio. The half-maximal concentration ( K 1/2) of SR ATPase was 335 nM Ca2+. The ATP synthesis dependence was similar with a K 1/2 of 243 nM for dehydrogenases and 114 nM for overall ATP production. In the reconstituted system, Ca2+ increased thapsigargin-sensitive ATP production (maximum ∼5-fold) with minimal changes in mitochondrial reduced nicotinamide adenine dinucleotide (NADH). NADH concentration remained stable despite graded increases in NADH turnover induced over a wide range of Ca2+ concentrations (0 to ∼500 nM). These data are consistent with a balanced activation of SR ATPase and mitochondrial ATP synthesis by Ca2+ that contributes to a homeostasis of energy metabolism metabolites. It is suggested that this balanced activation by cytosolic Ca2+ is partially responsible for the minimal alteration in energy metabolism intermediates that occurs with changes in cardiac workload in vivo.

scholarly journals The Role of Herbal Bioactive Components in Mitochondria Function and Cancer Therapy

2019 ◽  
Vol 2019 ◽  
pp. 1-12 ◽  
Author(s):  
Fangfang Tao ◽  
Yanrong Zhang ◽  
Zhiqian Zhang
Keyword(s):  
Cancer Therapy ◽  
Cancer Cell ◽  
Apoptotic Cell ◽  
Redox Status ◽  
Cancer Therapies ◽  
Bioactive Components ◽  
Atp Production

Mitochondria are highly dynamic double-membrane organelles which play a well-recognized role in ATP production, calcium homeostasis, oxidation-reduction (redox) status, apoptotic cell death, and inflammation. Dysfunction of mitochondria has long been observed in a number of human diseases, including cancer. Targeting mitochondria metabolism in tumors as a cancer therapeutic strategy has attracted much attention for researchers in recent years due to the essential role of mitochondria in cancer cell growth, apoptosis, and progression. On the other hand, a series of studies have indicated that traditional medicinal herbs, including traditional Chinese medicines (TCM), exert their potential anticancer effects as an effective adjunct treatment for alleviating the systemic side effects of conventional cancer therapies, for reducing the risk of recurrence and cancer mortality and for improving the quality of patients’ life. An amazing feature of these structurally diverse bioactive components is that majority of them target mitochondria to provoke cancer cell-specific death program. The aim of this review is to summarize the in vitro and in vivo studies about the role of these herbs, especially their bioactive compounds in the modulation of the disturbed mitochondrial function for cancer therapy.

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