1.A Novel In Vitro Method for the Metabolism Studies of Radiotracers Using Mouse Liver S9 Fraction.
Eun Kyoung RYU ; Yearn Seong CHOE ; Dong Hyun KIM ; Sang Yoon LEE ; Yong CHOI ; Kyung Han LEE ; Byung Tae KIM
Korean Journal of Nuclear Medicine 2004;38(4):325-329
PURPOSE: Usefulness of mouse liver S9 fraction was evaluated for the measurement of the metabolites in the in vitro metabolism study of 18F-labeled radiotracers. MATERIALS AND METHODS: Mouse liver S9 fraction was isolated at an early step in the course of microsome preparation. The in vitro metabolism studies were carried out by incubating a mixture containing the radiotracer, S9 fraction and NADPH at 37 degrees C, and an aliquot of the mixture was analyzed at the indicated time points by radio-TLC. Metabolic defluorination was further confirmed by the incubation with calcium phosphate, a bone mimic. RESULTS: The radiotracer [18F]1 underwent metabolic defluorination within 15 min, which was consistent with the results of the in vivo method and the in vitro method using microsome. Radiotracer [18F]2 was metabolized to three metabolites including 4-[18F]fluorobenzoic acid within 60 min. It is likely that the one of these metabolites at the origin of radio-TLC was identical with the one that obtained from the in vivo and in vitro (microsome) method. Compared with the in vitro method using microsome, the method using S9 fraction gave a similar pattern of the metabolites but with a different ratio, which can be explained by the presence of cytosol in the S9 fraction. CONCLUSION: These results suggest that the findings of the in vitro metabolism studies using S9 fraction can reflect the in vivo metabolism of novel radiotracers in the liver. Moreover, this method can be used as a tool to determine metabolic defluorination along with calcium phosphate absorption method.
Absorption
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Animals
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Calcium
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Cytosol
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Liver*
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Metabolism*
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Mice*
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Microsomes
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NADP
3.Relationship between hypoxia-induced apoptosis and caspases-3 activation, intracellular calcium overload in cardiomyocytes.
Zhou ZHOU ; Xiao-hua WANG ; Guang-xu ZHU ; Zheng-ping YU
Chinese Journal of Applied Physiology 2005;21(1):10-14
AIMTo explore the effects of hypoxia on Caspases activation in cardiomyocyte and role of intracellular calcium in this event in cardiomyocytes.
METHODSAfter hypoxia 0 min, 30 min, 1 h, 3 h, 6 h, 12 h, 24 h, apoptotic cell percentage was determined with Hoechst 33342 straining. Expressions of Caspases-3 mRNA and release of mitochondrial cytochrome c in primary culture of cardiomyocytes were determined by using RT-PCR and Western blotting respectively.
RESULTSElevation of Cyt c in cytosol was in accordance with the decline in mitochondrial Cyt c content. Significant increase in Cyt c in cytosol appeared at 12 h post hypoxia and peaked at 24 h while Cyt c in mitochondria could not be detected at 24 h post hypoxia. Hypoxia up-regulated Caspases-3 mRNA expressions beginning at 3 h post hypoxia. Intracellular calcium overload occurred earlier than release of mitochondrial Cyt c and the activation of Caspase-3 during the hypoxic insult. Inhibition of Caspase-3 activation and pretreatment with calcium chelator BAPTA/AM offered a marked protective effect on hypoxia induced cardiomyocyte apoptosis.
CONCLUSIONHypoxia can induce mitochondrion-dependent Caspase-3 activation in cardiomyocytes and therefore leads to cell apoptosis. Increase of intracellular Ca2+ plays an important role in the activation of Caspase-3 and the induction of apoptosis in cardiomyocytes.
Animals ; Apoptosis ; Calcium ; metabolism ; Caspase 3 ; metabolism ; Cell Hypoxia ; Cytochromes c ; metabolism ; Cytosol ; metabolism ; Male ; Mitochondria ; metabolism ; Myocytes, Cardiac ; cytology ; metabolism ; Rats ; Rats, Wistar
4.Cancer Energy Metabolism: Shutting Power off Cancer Factory
Biomolecules & Therapeutics 2018;26(1):39-44
In 1923, Dr. Warburg had observed that tumors acidified the Ringer solution when 13 mM glucose was added, which was identified as being due to lactate. When glucose is the only source of nutrient, it can serve for both biosynthesis and energy production. However, a series of studies revealed that the cancer cell consumes glucose for biosynthesis through fermentation, not for energy supply, under physiological conditions. Recently, a new observation was made that there is a metabolic symbiosis in which glycolytic and oxidative tumor cells mutually regulate their energy metabolism. Hypoxic cancer cells use glucose for glycolytic metabolism and release lactate which is used by oxygenated cancer cells. This study challenged the Warburg effect, because Warburg claimed that fermentation by irreversible damaging of mitochondria is a fundamental cause of cancer. However, recent studies revealed that mitochondria in cancer cell show active function of oxidative phosphorylation although TCA cycle is stalled. It was also shown that blocking cytosolic NADH production by aldehyde dehydrogenase inhibition, combined with oxidative phosphorylation inhibition, resulted in up to 80% decrease of ATP production, which resulted in a significant regression of tumor growth in the NSCLC model. This suggests a new theory that NADH production in the cytosol plays a key role of ATP production through the mitochondrial electron transport chain in cancer cells, while NADH production is mostly occupied inside mitochondria in normal cells.
Adenosine Triphosphate
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Aldehyde Dehydrogenase
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Cytosol
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Electron Transport
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Energy Metabolism
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Fermentation
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Glucose
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Lactic Acid
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Metabolism
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Mitochondria
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NAD
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Oxidative Phosphorylation
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Oxygen
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Symbiosis
5.Effect of hypothermic cardioplegia on cardiac protection--I. Effect of hypothermic cardioplegia on the cytosolic Ca2+ concentration in rat ventricular myocytes.
Duck Sun AHN ; Young Ho LEE ; Doo Hee KANG ; Bok Soon KANG
Yonsei Medical Journal 1994;35(2):162-176
Cytosolic Ca2+ concentration of rat ventricular cells was measured under varying experimental conditions by using a fluorescent Ca2+ indicator, Fura-2. Resting [Ca2+]i of rat myocyte was 150 +/- 30 nM (n = 39), and this value was compatible with others. The Perfusion of cardioplegic solution significantly increased [Ca2+]i, and this effect was further augmented by hypothermia (p<0.05). Application of nifedipine (5 x 10(-7) M) to the perfusate or pretreatment of caffeine (10 mM) had no apparent effect on this cardioplegia-induced [Ca2+]i change. But Ni2+ (5 mM), an antagonist of Na+/Ca2+ exchange mechanism, prevented the [Ca2+]i change during cardioplegia (p<0.05). Magnitude of cardioplegia-induced [Ca2+]i increase was also dependent on the Ca2+ concentration of cardioplegic solution. These results suggest that Na+/Ca2+ exchange may play an important role in cardioplegia-induced [Ca2+]i change. To rule out the possibility whether the protective effect of hypothermic cardioplegia is due to the preservation of high-energy phosphate store or decreasing the transmembrane ionic fluxes by phase transition, we exhausted a energy store of cardiac cell by application of 2,4 dinitrophenol to the bath and measured its effect on [Ca2+]i change during cardioplegia. Hypothermic cardioplegia delayed the onset of [Ca2+]i increase and decreased its amplitude compared to those of normothermic cardioplegia. From the above results, hypothermic cardioplegia may protect the cardiac cells from ischemic insult by preserving a high-energy phosphate store. Application of Ni2+ to the cardioplegic solution or reduction of external Ca2+ concentration also had some protective effect, since it prevented [Ca2+]i increase during cardioplegia.
Animal
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Calcium/*metabolism
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Cytosol/metabolism
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*Heart Arrest, Induced
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Heart Ventricle/metabolism
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Hypothermia, Induced
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Myocardial Ischemia/metabolism/*prevention & control
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Myocardium/*metabolism
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Rats
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Support, Non-U.S. Gov't
6.The cytosolic domain of Bcl-2 oligomerizes to form pores in model mitochondrial outer membrane at acidic pH.
Jun PENG ; Suzanne M LAPOLLA ; Zhi ZHANG ; Jialing LIN
Journal of Biomedical Engineering 2009;26(3):631-637
The three dimensional structures of both pro-apoptotic Bax and anti-apoptotic Bcl-2 are strikingly similar to that of pore-forming domains of diphtheria toxin and E. coli colicins. Consistent with the structural similarity, both Bax and Bcl-2 have been shown to possess pore-forming property in the membrane. However, these pore-forming proteins form pores via different mechanisms. While Bax and diphtheria toxin form pores via oligomerization, the colicin pore is formed only by colicin monomers. Although the oligomers of Bcl-2 proteins have been found in the mitochondria of both healthy and apoptotic cells, it is unknown whether or not oligomerization is involved in the pore formation. To determine the mechanism of Bcl-2 pore formation, we reconstituted the pore-forming process of Bcl-2 using purified proteins and liposomes. We found that Bcl-2 pore size depended on Bcl-2 concentration, and the release of smaller entrapped molecules was faster than that of larger ones from liposomes at a given Bcl-2 concentration. Moreover, the rate of dye release mediated by pre-formed wild-type Bcl-2 oligomers or by the mutant Bcl-2 monomers with a higher homo-association affinity was much higher than that by wild-type Bcl-2 monomers. Together, it is suggested that oligomerization is likely involved in Bcl-2 pore formation.
Apoptosis
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physiology
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Cytosol
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metabolism
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Humans
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Hydrogen-Ion Concentration
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Liposomes
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metabolism
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Mitochondrial Membrane Transport Proteins
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metabolism
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Mitochondrial Membranes
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metabolism
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Protein Multimerization
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Proto-Oncogene Proteins c-bcl-2
;
metabolism
7.Neuronal modeling with intracellular calcium signaling.
Acta Physiologica Sinica 2011;63(5):442-452
Cytosolic Ca(2+) ions play an important role in the regulation of numerous aspects of cellular activity in virtually all cell types. There is a complex interaction between the neuronal electrical signals on plasma membrane and the chemical signals of intracellular calcium. Each neuron can be considered as a binary membrane system with plasma membrane and endoplasmic reticulum membrane, and the neuronal endoplasmic reticulum can be regarded as a neuron-within-a-neuron. This review explores the simulation modeling of neuronal dynamics mutually coupled with the intracellular calcium signaling released from endoplasmic reticulum through the inositol 1,4,5-trisphosphate receptor calcium channels. We show that a current trend is to include the intracellular calcium dynamics into the neuronal models, and the frontier of this research is now shifting to spatial neuronal models with diffusing intracellular calcium. It is expected that more important results will be obtained with the neuronal models incorporating the intracellular calcium dynamics, especially the spatial models considering the calcium diffusion both in soma and dendritic branches.
Animals
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Calcium
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metabolism
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Calcium Signaling
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physiology
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Cytosol
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metabolism
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Endoplasmic Reticulum
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metabolism
;
physiology
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Humans
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Inositol 1,4,5-Trisphosphate Receptors
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metabolism
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Models, Neurological
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Neurons
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metabolism
;
physiology
8.Effects of tetrandrine on cytosolic free calcium concentration in corpus cavernosum smooth muscle cells of rabbits.
Ji-Hong LIU ; Jun CHEN ; Tao WANG ; Bo LIU ; Jun YANG ; Xiao-Wen CHEN ; Shao-Gang WANG ; Chun-Ping YIN ; Zhang-Qun YE
Asian Journal of Andrology 2006;8(4):405-409
AIMTo study the relaxation mechanisms of tetrandrine (Tet) on the corpus cavernosum smooth muscle.
METHODSThe corpus cavernosum smooth muscle cells from New Zealand white rabbits were cultured in vitro. [Ca(2+)](i) was measured by Fluorescence Ion Digital Imaging System, using Fluo-2/AM as a Ca(2+)-sensitive fluorescent indicator.
RESULTSTet (1, 10 and 100 micromol/L) had no effect on the resting [Ca(2+)](i) (P>0.05). In the presence of extracellular Ca(2+) (2.5 mmol/L), Tet (1, 10 and 100 micromol/L) inhibited [Ca(2+)](i) elevation induced by high K(+) and phenylephrine (PE) in a concentration-dependent manner (P>0.05). In calcium free solution containing egtaic acid, Tet (1 and 10 micromol/L) had no inhibitory effects on [Ca(2+)](i) elevation induced by PE (P>0.05). However, Tet (100 micromol/L) inhibited [Ca(2+)](i) elevation induced by PE (P>0.05).
CONCLUSIONTet inhibited the Ca(2+) influx from the extracellular site via voltage-activated Ca(2+) channel and alpha(2)-adrenoceptor-operated Ca(2+) channel. At a high concentration, Tet might inhibit the cytosolic calcium pool release in cultured corpus cavernosum smooth muscle cells. This inhibitory action on [Ca(2+)](i) might be one of the relaxation mechanisms of Tet on the corpus cavernosum smooth muscle.
Alkaloids ; pharmacology ; Animals ; Benzylisoquinolines ; pharmacology ; Calcium ; metabolism ; Cytosol ; drug effects ; metabolism ; Male ; Muscle Relaxation ; Muscle, Smooth, Vascular ; cytology ; metabolism ; Penis ; cytology ; metabolism ; Phenylephrine ; pharmacology ; Potassium Chloride ; pharmacology ; Rabbits
9.Structural characterization of coatomer in its cytosolic state.
Shengliu WANG ; Yujia ZHAI ; Xiaoyun PANG ; Tongxin NIU ; Yue-He DING ; Meng-Qiu DONG ; Victor W HSU ; Zhe SUN ; Fei SUN
Protein & Cell 2016;7(8):586-600
Studies on coat protein I (COPI) have contributed to a basic understanding of how coat proteins generate vesicles to initiate intracellular transport. The core component of the COPI complex is coatomer, which is a multimeric complex that needs to be recruited from the cytosol to membrane in order to function in membrane bending and cargo sorting. Previous structural studies on the clathrin adaptors have found that membrane recruitment induces a large conformational change in promoting their role in cargo sorting. Here, pursuing negative-stain electron microscopy coupled with single-particle analyses, and also performing CXMS (chemical cross-linking coupled with mass spectrometry) for validation, we have reconstructed the structure of coatomer in its soluble form. When compared to the previously elucidated structure of coatomer in its membrane-bound form we do not observe a large conformational change. Thus, the result uncovers a key difference between how COPI versus clathrin coats are regulated by membrane recruitment.
ADP-Ribosylation Factor 1
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chemistry
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metabolism
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Animals
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Coatomer Protein
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chemistry
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metabolism
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Cytosol
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chemistry
;
metabolism
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GTPase-Activating Proteins
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chemistry
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metabolism
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Humans
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Membranes, Artificial
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Rats
10.Defective Mitochondrial Function and Motility Due to Mitofusin 1 Overexpression in Insulin Secreting Cells.
Kyu Sang PARK ; Andreas WIEDERKEHR ; Claes B WOLLHEIM
The Korean Journal of Physiology and Pharmacology 2012;16(1):71-77
Mitochondrial dynamics and distribution is critical for their role in bioenergetics and cell survival. We investigated the consequence of altered fission/fusion on mitochondrial function and motility in INS-1E rat clonal beta-cells. Adenoviruses were used to induce doxycycline-dependent expression of wild type (WT-Mfn1) or a dominant negative mitofusin 1 mutant (DN-Mfn1). Mitochondrial morphology and motility were analyzed by monitoring mitochondrially-targeted red fluorescent protein. Adenovirus-driven overexpression of WT-Mfn1 elicited severe aggregation of mitochondria, preventing them from reaching peripheral near plasma membrane areas of the cell. Overexpression of DN-Mfn1 resulted in fragmented mitochondria with widespread cytosolic distribution. WT-Mfn1 overexpression impaired mitochondrial function as glucose- and oligomycin-induced mitochondrial hyperpolarization were markedly reduced. Viability of the INS-1E cells, however, was not affected. Mitochondrial motility was significantly reduced in WT-Mfn1 overexpressing cells. Conversely, fragmented mitochondria in DN-Mfn1 overexpressing cells showed more vigorous movement than mitochondria in control cells. Movement of these mitochondria was also less microtubule-dependent. These results suggest that Mfn1-induced hyperfusion leads to mitochondrial dysfunction and hypomotility, which may explain impaired metabolism-secretion coupling in insulin-releasing cells overexpressing Mfn1.
Adenoviridae
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Animals
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Cell Membrane
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Cell Survival
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Cytosol
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Energy Metabolism
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Insulin
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Insulin-Secreting Cells
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Luminescent Proteins
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Mitochondria
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Mitochondrial Dynamics
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Rats