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            P nuclear magnetic resonance studies of HeLa cells

Evans, Frederick E.; Kaplan, Nathan O.

doi:10.1073/pnas.74.11.4909

Cited by 63 publications

(22 citation statements)

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“…The most prominent peaks are due to phosphocholine, inorganic phosphate (P i ), the ␥-phosphate of nucleoside triphosphate (␥-NTP) (which also includes ␤-NDP), ␣-NTP (which also includes ␣-NDP), ␤-NTP, and uridine diphosphate sugars (UDPS). The assignment of the peaks was based on known chemical shifts of phosphate metabolites from previous studies (17,18), and by spiking the NMR spectra of uninfected HeLa cell extracts with uridine 5Ј-diphospho-N-acetylglucosamine, uridine 5Ј-diphosphoglucoronic acid, and uridine 5Ј-diphosphoglucose. A typical extract spectrum is shown in Fig.…”

Section: Growth and Infection Of Hela Cells Onmentioning

confidence: 99%

Enhancement of ATP Levels and Glucose Metabolism during an Infection by Chlamydia

Ojcius¹,

Degani²,

Mispelter³

et al. 1998

Journal of Biological Chemistry

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The Chlamydia species are obligate intracellular bacteria that proliferate only within the infected cell. Since the extracellular bacteria are metabolically inert and there are no cell-free systems for characterizing Chlamydia metabolism, we studied metabolic changes related to ATP synthesis and glycolysis in HeLa cells infected with Chlamydia psittaci during the course of the 2-day infection cycle using noninvasive 31 P and 13 C NMR methods. We find that the infection stimulates ATP synthesis in the infected cell, with a peak of ATP levels occurring midway through the infection cycle, when most of the metabolically active bacteria are proliferating. The infection also stimulates synthesis of glutamate with a similar time course as for ATP. The stimulation is apparently due to an enhancement in glucose consumption by the infected cell, which also results in an increased rate of lactate production and glutamate synthesis as well as higher glycogen accumulation during the infection. Concurrently, infection leads to an increase in the expression of the glucose transporter, GLUT-1, on HeLa cells, which may account for the enhanced glucose consumption. The chlamydiae are thus able to stimulate glucose transport in the host cell sufficiently to compensate for the extra energy load on the cell represented by the infection.Chlamydia species are causative agents of conjunctivitis and pneumonia, and they are recognized as the leading cause of bacterially acquired sexually transmitted infections. On repeated exposure, the consequences can be blinding trachoma or sequelae from sexually transmitted infection, such as epididymitis or pelvic inflammatory diseases, ectopic pregnancy, or tubal infertility (1, 2). Despite the clinical importance of these infections, the biology and biochemistry of Chlamydia infection remains poorly understood.The chlamydiae are obligate intracellular bacteria that proliferate only within the infected host cell. In line with its requirement for host cell metabolites for survival, Chlamydia exists in two developmental states, elementary bodies (EBs) 1 and reticulate bodies (RBs). EBs represent the metabolically inactive, infectious form of the bacteria, and they are internalized into host cells within membrane-bound vacuoles that avoid fusion with host-cell lysosomes (3). Within 6 -10 h after internalization, the EBs differentiate into the metabolically active RBs. This transformation triggers DNA, RNA, and protein synthesis in the RBs, which proliferate in the growing vacuole and produce up to a thousand progeny. Lipids from the transGolgi network are transported to this membrane (4), and the bacteria contribute their own proteins to the inclusion membrane (5). An intimate association thus exists between the bacteria and the host, but the lack of molecular tools in the field has made further characterization of the association difficult. After approximately 2 days of infection, RBs differentiate back into EBs, and a new infection cycle can begin.Chlamydia trachomatis grows well in cytoplasts (enuc...

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Section: Growth and Infection Of Hela Cells Onmentioning

confidence: 99%

Enhancement of ATP Levels and Glucose Metabolism during an Infection by Chlamydia

Ojcius¹,

Degani²,

Mispelter³

et al. 1998

Journal of Biological Chemistry

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“…Distinct from earlier 31 P MR spectroscopy (MRS) studies that reported total NAD contents (18)(19)(20), our approach is able to resolve the MR signal of NADH from that of NAD + by taking advantage of their specific spectroscopic characteristics at a given magnetic field strength that can be precisely predicted based on a theoretical model (17). Thus, both NAD + and NADH can be quantified simultaneously by matching the in vivo NAD spectra with the model-simulated spectra.…”

mentioning

confidence: 99%

In vivo NAD assay reveals the intracellular NAD contents and redox state in healthy human brain and their age dependences

Zhu

Lü

Lee

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

386

347

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NAD is an essential metabolite that exists in NAD + or NADH form in all living cells. Despite its critical roles in regulating mitochondrial energy production through the NAD + /NADH redox state and modulating cellular signaling processes through the activity of the NAD + -dependent enzymes, the method for quantifying intracellular NAD contents and redox state is limited to a few in vitro or ex vivo assays, which are not suitable for studying a living brain or organ. Here, we present a magnetic resonance (MR) -based in vivo NAD assay that uses the high-field MR scanner and is capable of noninvasively assessing NAD + and NADH contents and the NAD + /NADH redox state in intact human brain. The results of this study provide the first insight, to our knowledge, into the cellular NAD concentrations and redox state in the brains of healthy volunteers. Furthermore, an age-dependent increase of intracellular NADH and age-dependent reductions in NAD + , total NAD contents, and NAD + /NADH redox potential of the healthy human brain were revealed in this study. The overall findings not only provide direct evidence of declined mitochondrial functions and altered NAD homeostasis that accompany the normal aging process but also, elucidate the merits and potentials of this new NAD assay for noninvasively studying the intracellular NAD metabolism and redox state in normal and diseased human brain or other organs in situ.redox state | NAD | in vivo 31 P MR spectroscopy | human brain | aging N AD, a multifunctional metabolite found in all living cells, has been the interest of many scientific investigations since its discovery in the early 20th century (1). NAD is known to convert between its oxidized NAD + and reduced NADH forms during the breakdown of nutrients; hence, the intracellular NAD + /NADH redox state reflects the metabolic balance of the cell in generating ATP energy through oxidative phosphorylation in mitochondria and/or glycolysis in cytosol (2). More recently, after several protein families associated with cell survival were found to use NAD + as their main substrate with activities also regulated by the availability of the NAD + , the full extent of the NAD's function as a metabolic regulator began to unfold (3-5). A growing number of studies have indicated that NAD + can modulate metabolic signaling pathways and mediate important cellular processes, including calcium homeostasis, gene expression, aging, degeneration, and cell death; therefore, the cellular NAD could serve as a therapeutic target for treating various metabolic or age-related diseases and promoting longevity (6-12).Despite the critical relevance of the intracellular NAD metabolism to human health and diseases, assessment of NAD contents and NAD + /NADH redox state is extremely challenging. Only a few invasive techniques based on biochemical assays or autofluorescence methods have been used to analyze tissue samples or cell extracts (13,14). However, during the preparation of such ex vivo sample, the NAD + and NADH contents are likely altered, beca...

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“…Expanding the spectra revealed another triphosphate near the P resonances of ATP (11) that accounted for 25 ± 4% (s.d., n = 3) of the total peak area in GLC4/ADR extracts and for 19 ± 4% in GLC4 extracts. This triphosphate could be UTP, GTP or CTP (Evans & Kaplan, 1977;Evanochko et al, 1984). High resolution 'H NMR spectra were obtained from extracts of both cell lines in D20.…”

Section: Resultsmentioning

confidence: 99%

“…Assignments were made on basis of data in the literature (Daly et al, 1987;Evans & Kaplan, 1977;Evanochko et al, 1984) and by adding standard compounds. Extracts of both cell lines showed high levels of PC (1 in Figure la and b).…”

Section: Resultsmentioning

confidence: 99%

“…Ice-cold perchloric acid (10%) was added to the pellet and the cell mixture was vortexed at the beginning and the end of a 20 min period. The extracts were neutralised with KOH, centrifuged to remove the KC104 precipitate, freeze-dried and redissolved in D20 (Evans & Kaplan, 1977 Since the relative separations between the P and a, and the and peaks of ATP are proportional to the amount of ATP bound to Mg2+, the fraction of total ATP that is not complexed to Mg2+ (0) can be calculated (Gupta & Moore, 1980a). The free Mg2+ concentration can then be calculated using the dissociation constant of MgATP (ATP complexed to magnesium) (Kd= 38 gM) and the formula [Mg2]= Kd (0'-1 ) (Gupta & Moore, 1980a).…”

Section: Methodsmentioning

confidence: 99%

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NMR spectroscopy analysis of phosphorus metabolites and the effect of adriamycin on these metabolite levels in an adriamycin-sensitive and -resistant human small cell lung carcinoma cell line

Jong

Mulder²,

Vries

et al. 1991

Br J Cancer

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Summary 3'P nuclear magnetic resonance (NMR) spectra of cells and of cell extracts revealed high levels of phosphorylcholine (PC) and phosphocreatine (PCr) in an adriamycin-resistant human small cell lung carcinoma cell line (GLC4/ADR) and the adriamycin-sensitive parental cell line (GLC4). PCr levels in extracts of GLC4/ADR were increased compared to extracts of GLC4. We estimated that 11% of the total intracellular ATP is not bound to Mg2" in both cell lines. This value corresponded to an intracellular free Mg2" of 0.30 mM. The effects of different adriamycin concentrations, 0.05, 1 and 30 JLM for GLC4 and 1, 30 and 200 LM for GLC4/ADR, on the phosphorus metabolite levels in continuously perfused cells were monitored.Significant differences between GLC4 and GLC4/ADR included: (a) a strong increase in the PATP level in the presence of 30 tLM adriamycin in GLC4 only, followed by a fast decrease after 5 h of perfusion. (b) a less dramatic increase in the PC level in GLC4/ADR and an unchanged ATP level in the presence of increasing adriamycin concentrations. (c) an increased GPC level in GLC4/ADR in the presence of adriamycin. The changes in PC and GPC levels in the presence of adriamycin suggested that the phospholipid turnover was increased in GLC4/ADR and could be stimulated in the presence of adriamycin. In both cell lines, PCr levels decreased faster than the ATP levels after adriamycin treatment. Thus, biochemical markers for adriamycin resistance can be detected with NMR spectroscopy. However, more studies are necessary to obtain parameters to distinguish drug-sensitive from drug-resistant tumours in patients by NMR spectroscopy.

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³¹ P nuclear magnetic resonance studies of HeLa cells

Cited by 63 publications

References 15 publications

Enhancement of ATP Levels and Glucose Metabolism during an Infection by Chlamydia

Enhancement of ATP Levels and Glucose Metabolism during an Infection by Chlamydia

In vivo NAD assay reveals the intracellular NAD contents and redox state in healthy human brain and their age dependences

NMR spectroscopy analysis of phosphorus metabolites and the effect of adriamycin on these metabolite levels in an adriamycin-sensitive and -resistant human small cell lung carcinoma cell line

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31 P nuclear magnetic resonance studies of HeLa cells

Cited by 63 publications

References 15 publications

Enhancement of ATP Levels and Glucose Metabolism during an Infection by Chlamydia

Enhancement of ATP Levels and Glucose Metabolism during an Infection by Chlamydia

In vivo NAD assay reveals the intracellular NAD contents and redox state in healthy human brain and their age dependences

NMR spectroscopy analysis of phosphorus metabolites and the effect of adriamycin on these metabolite levels in an adriamycin-sensitive and -resistant human small cell lung carcinoma cell line

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³¹ P nuclear magnetic resonance studies of HeLa cells