RNA cytosine methyltransferase Nsun3 regulates embryonic stem cell differentiation by promoting mitochondrial activity

Chemical modifications of RNA have been attracting increasing interest because of their impact on RNA fate and function. Therefore, the characterization of enzymes catalyzing such modifications is of great importance. The RNA cytosine methyltransferase NSUN3 was recently shown to generate 5-methylcytosine in the anticodon loop of mitochondrial tRNA^Met. Further oxidation of this position is required for normal mitochondrial translation and function in human somatic cells. Because embryonic stem cells (ESCs) are less dependent on oxidative phosphorylation than somatic cells, we examined the effects of catalytic inactivation of Nsun3 on self-renewal and differentiation potential of murine ESCs. We demonstrate that Nsun3-mutant cells show strongly reduced mt-tRNA^Met methylation and formylation as well as reduced mitochondrial translation and respiration. Despite the lower dependence of ESCs on mitochondrial activity, proliferation of mutant cells was reduced, while pluripotency marker gene expression was not affected. By contrast, ESC differentiation was skewed towards the meso- and endoderm lineages at the expense of neuroectoderm. Wnt3 was overexpressed in early differentiating mutant embryoid bodies and in ESCs, suggesting that impaired mitochondrial function disturbs normal differentiation programs by interfering with cellular signalling pathways. Interestingly, basal levels of reactive oxygen species (ROS) were not altered in ESCs, but Nsun3 inactivation attenuated induction of mitochondrial ROS upon stress, which may affect gene expression programs upon differentiation. Our findings not only characterize Nsun3 as an important regulator of stem cell fate but also provide a model system to study the still incompletely understood interplay of mitochondrial function with stem cell pluripotency and differentiation.     

Mitochondrial calcium efflux and porcine stress-susceptibility

Summary Mitochondrial Ca²⁺ efflux rates ofM. longissimus dorsi correlate very closely with parameters associated with porcine stress-susceptibility. Experimental data support the measurement of mitochondrial Ca²⁺ efflux to be a very sensitive and reliable method for differentiating porcine stress-susceptibility.Acknowledgments. The autors are grateful to Dr Webb (Edinburgh) and to Dr Monin (Theix) for supplying halothanescreened pigs, to Mr C.C. Ketteridge for technical assistance and to Mr N. F. Doun for drip measurements.     

Impact of atypical mitochondrial cyclic-AMP level in nephropathic cystinosis

Nephropathic cystinosis (NC) is a rare disease caused by mutations in the CTNS gene encoding for cystinosin, a lysosomal transmembrane cystine/H⁺ symporter, which promotes the efflux of cystine from lysosomes to cytosol. NC is the most frequent cause of Fanconi syndrome (FS) in young children, the molecular basis of which is not well established. Proximal tubular cells have very high metabolic rate due to the active transport of many solutes. Not surprisingly, mitochondrial disorders are often characterized by FS. A similar mechanism may also apply to NC. Because cAMP has regulatory properties on mitochondrial function, we have analyzed cAMP levels and mitochondrial targets in CTNS^?/? conditionally immortalized proximal tubular epithelial cells (ciPTEC) carrying the classical homozygous 57-kb deletion (delCTNS^?/?) or with compound heterozygous loss-of-function mutations (mutCTNS^?/?). Compared to wild-type cells, cystinotic cells had significantly lower mitochondrial cAMP levels (delCTNS^?/? ciPTEC by 56%?±?10.5, P?<?0.0001; mutCTNS^?/? by 26%?±?4.3, P?<?0.001), complex I and V activities, mitochondrial membrane potential, and SIRT3 protein levels, which were associated with increased mitochondrial fragmentation. Reduction of complex I and V activities was associated with lower expression of part of their subunits. Treatment with the non-hydrolysable cAMP analog 8-Br-cAMP restored mitochondrial potential and corrected mitochondria morphology. Treatment with cysteamine, which reduces the intra-lysosomal cystine, was able to restore mitochondrial cAMP levels, as well as most other abnormal mitochondrial findings. These observations were validated in CTNS-silenced HK-2 cells, indicating a pivotal role of mitochondrial cAMP in the proximal tubular dysfunction observed in NC.     

Calcium and sodium distribution and movements in smooth muscle

Summary Electron probe microanalysis (EPMA) has been used to study the subcellular distribution of Ca, Na, K. Cl, and Mg in smooth muscle. The EPMA results indicate that the sarcoplasmic reticulum (SR) is the majorintracellular source and sink of activator Ca: norepinephrine decreases the Ca content of the junctional SR in portal vein smooth muscle. Mitochondria do not play a significant role in regulating cytoplasmic free Ca²⁺, but mitochondrial Ca content can be altered to a degree compatible with suggestions that fluctuations in matrix Ca contribute to the control of mitochondrial metabolism. The rise intotal cytoplasmic Ca during a maintained, maximal contraction is very much greater than the rise in free Ca²⁺, and is probably in excess of the known binding sites available on calmodulin and myosin. Cell Ca is not increased in normal cells that are Na-loaded. The non-Donnan distribution of Cl is not due to compartmentalization, but reflects high cytoplasmic Cl. Na-loading of smooth muscle in K-free solutions is temperature dependent, and may exhibit cellular heterogeneity undetected by conventional techniques. The total cell Mg is equivalent to approximately 12 mM, and less than 50% of it can be accounted for by binding to ATP and to actin. Mitochondrial monovalent cations in smooth muscle are relatively rapidly exchangeable.     

Hypotensive activity of histidine-containing analogues of C-terminal hexapeptide of Substance P

Summary Four new hexapeptide analogues of C-terminal Substance P fragment with increased solubility in aqueous solutions are described. The peptides contain histidine in positions 6, 8, 9 and 10, respectively. The effect of the structural changes on the hypotensive activity and antigenic properties of analogues was compared. It was found that substitution of amino acid residues in various positions in the C-terminal hexapeptide of Substance P resulted in different effects on the hypotensive and antigenic properties, respectively. Only the [His⁶] SP_6-11 analogue had an unchanged antigenic structure when compared with the C-terminal region of Substance P, but it showed an almost total loss of hypotensive activity. The [His⁹] SP_6-11 analogue retained 50% of the hypotensive activity of the C-terminal hexapeptide but showed a markedly reduced expression of the antigenic epitope localized in this region of Substance P.     

Protective effect of N-(2-propynyl)-2-(5-benzyloxy-indolyl) methylamine (PF9601N) on mitochondrial permeability transition

PF9601N, N-(2-propynyl)-2-(5-benzyloxy-indolyl) methylamine, an monoamine oxidase (MAO) B inhibitor, has shown neuroprotective properties against dopaminergic toxins. To elucidate the mechanisms involved in this protection, the effect of PF9601N on mitochondria was assessed. PF9601N prevents mitochondrial swelling, drop in the electrical potential and oxidation of sulfhydryl groups, glutathione and pyridine nucleotides induced by Ca²⁺. These observations demonstrate the protective effect of PF9601N on the induction of mitochondrial permeability transition. This protection is due to the interaction of the secondary protonated amino group in the molecule with pore-forming structures and to its antioxidant property, rather than to inhibition of MAO B activity. PF9601N also prevents the release of cytochrome c from mitochondria, suggesting its potential inhibitory effect on mitochondria-mediated apoptosis. The low IC⁵⁰ value for this inhibition, in comparison with deprenyl, make it a more efficient compound than propargylamines and other amines in protecting the bioenergetic functions of mitochondria. Received 9 March 2006; received after revision 10 April 2006; accepted 21 April 2006     

Isoform specific phosphorylation of p53 by protein kinase CK1

The ability of three isoforms of protein kinase CK1 (α, γ₁, and δ) to phosphorylate the N-terminal region of p53 has been assessed using either recombinant p53 or a synthetic peptide reproducing its 1–28 sequence. Both substrates are readily phosphoylated by CK1δ and CK1α, but not by the γ isoform. Affinity of full size p53 for CK1 is 3 orders of magnitude higher than that of its N-terminal peptide (K _m 0.82 μM vs 1.51 mM). The preferred target is S20, whose phosphorylation critically relies on E17, while S6 is unaffected despite displaying the same consensus (E-x-x-S). Our data support the concept that non-primed phosphorylation of p53 by CK1 is an isoform-specific reaction preferentially affecting S20 by a mechanism which is grounded both on a local consensus and on a remote docking site mapped to the K²²¹RQK²²⁴ loop according to modeling and mutational analysis.     

Dual antiglioma action of metformin: cell cycle arrest and mitochondria-dependent apoptosis

The present study reports for the first time a dual antiglioma effect of the well-known antidiabetic drug metformin. In low-density cultures of the C6 rat glioma cell line, metformin blocked the cell cycle progression in G₀/G₁ phase without inducing significant cell death. In confluent C6 cultures, on the other hand, metformin caused massive induction of caspase-dependent apoptosis associated with c-Jun N-terminal kinase (JNK) activation, mitochondrial depolarization and oxidative stress. Metformin-triggered apoptosis was completely prevented by agents that block mitochondrial permeability transition (cyclosporin A) and oxygen radical production (N-acetylcisteine), while the inhibitors of JNK activation (SP600125) or glycolysis (sodium fluoride, iodoacetate) provided partial protection. The antiglioma effect of metformin was reduced by compound C, an inhibitor of AMP-activated protein kinase (AMPK), and was mimicked by the AMPK agonist AICAR. Similar effects were observed in the human glioma cell line U251, while rat primary astrocytes were completely resistant to the antiproliferative and proapoptotic action of metformin. Received 14 February 2007; received after revision 26 March 2007; accepted 3 April 2007     

Nuclear localization of mouse fibroblast growth factor 2 requires N-terminal and C-terminal sequences

In vertebrates, different isoforms of fibroblast growth factor 2 (FGF2) exist, which differ by their N-terminal extension. They show different localization and expression levels and exert distinct biological effects. Nevertheless, genetic inactivation of all FGF2 isoforms in the mouse results in only mild phenotypes. Here, we analyzed mouse FGF2, and show that, as in the human, mouse FGF2 contains CTG-initiated high molecular-weight (HMW) isoforms, which contain a nuclear localization signal, and which mediate localization of this isoform to the nucleus. Using green fluorescent protein-FGF2 fusions, we furthermore observed, that C-terminal deletions disable nuclear localization of the short low-molecular-weight (LMW) 18-kDa isoform. This loss of specific localization is accompanied by a loss in heparin binding. We therefore suggest that, first, localization of mouse FGF2 is comparable to that in other vertebrates and, second, FGF2 contains at least two sequences important for nuclear localization, a nuclear localization sequence at the N terminus which is only contained in the HMW isoform, and another sequence at the C terminus, which is only required for localization of the LMW 18-kDa isoform. Received 1 July 2003; accepted 14 August 2003     

Dual modes of 5-(N-ethyl-N-isopropyl)amiloride modulation of apical dipeptide uptake in the human small intestinal epithelial cell line Caco-2

Selective pharmacological Na⁺/H⁺ exchange (NHE) inhibitors were used to identify functional NHE isoforms in human small intestinal enterocytes (Caco-2) and to distinguish between direct and indirect effects on transport via the intestinal di/tripeptide transporter hPepT1. The relative potencies of these inhibitors to inhibit ²²Na⁺ influx identifies NHE3 and NHE1 as the apical and basolateral NHE isoforms. The Na⁺-dependent (NHE3-sensitive) component of apical dipeptide ([¹⁴C] Gly-Sar) uptake was inhibited by the selective NHE inhibitors with the same order of potency observed for inhibition of apical ²²Na⁺ uptake. However, 5-(N-ethyl-N-isopropyl) amiloride (EIPA) also reduced [¹⁴C]Gly-Sar uptake in the absence of Na⁺ and this inhibition was concentration and pH (maximal at pH 5.5) dependent. NHE3 inhibition by S1611 and S3226 modulates dipeptide uptake indirectly by reducing the transapical driving force (H⁺ electrochemical gradient). EIPA (at 100 μM) has similar effects, but at higher concentrations (>200 μM) also has direct inhibitory effects on hPepT1.Received 28 February 2005; received after revision 20 April 2005; accepted 20 May 2005     

Differential water permeability and regulation of three aquaporin 4 isoforms

Aquaporin 4 (AQP4) is expressed in the perivascular glial endfeet and is an important pathway for water during formation and resolution of brain edema. In this study, we examined the functional properties and relative unit water permeability of three functional isoforms of AQP4 expressed in the brain (M1, M23, Mz). The M23 isoform gave rise to square arrays when expressed in Xenopus laevis oocytes. The relative unit water permeability differed significantly between the isoforms in the order of M1 > Mz > M23. None of the three isoforms were permeable to small osmolytes nor were they affected by changes in external K⁺ concentration. Upon protein kinase C (PKC) activation, oocytes expressing the three isoforms demonstrated rapid reduction of water permeability, which correlated with AQP4 internalization. The M23 isoform was more sensitive to PKC regulation than the longer isoforms and was internalized significantly faster. Our results suggest a specific role for square array formation.     

Vacuolar H+-ATPase: From mammals to yeast and back

Vacuolar H⁺-adenosine triphosphatase (V-ATPase) is composed of distinct catalytic (V₁) and membrane (V₀) sectors containing several subunits. The biochemistry of the enzyme was mainly studied in organelles from mammalian cells such as chromaffin granules and clathrin-coated vesicles. Subsequently, mammalian cDNAs and yeast genes encoding subunits of V-ATPase were cloned and sequenced. The sequence information revealed the relation between V- and F-ATPases that evolved from a common ancestor. The isolation of yeast genes encoding subunits of V-ATPase opened an avenue for molecular biology studies of the enzyme. Because V-ATPase is present in every known eukaryotic cell and provides energy for vital transport systems, it was anticipated that disruption of genes encoding V-ATPase subunits would be lethal. Fortunately, yeast cells can survive the absence of V-ATPase by drinking the acidic medium. So far only yeast cells have been shown to be viable without an active V-ATPase. In contrast to yeast, mammalian cells may have more than one gene encoding each of the subunits of the enzyme. Some of these genes encode tissue- and/or organelle-specific subunits. Expression of these specific cDNAs in yeast cells may reveal their unique functions in mammalian cells. Following the route from mammals to yeast and back may prove useful in the study of many other complicated processes.     

Effects of high hydrostatic pressures on the movements of Na+, K+ and Cl− in isolated eel gills

Summary Hydrostatic pressure applied to isolated eel gills induces changes in the tissue, Na⁺, K⁺ and Cl^– contents. It also inhibits the activity of the (Na⁺+K⁺) ATPase. Results are discussed in terms of an effect of pressure on the Na⁺ and Cl^– pumps and on the passive permeability processes.A. P., Chargé de Recherches du F. N. R. S.; R. G., Chercheur qualifié du F. N. R. S., We are grateful to Prof. A. Distèche and E. Schoffeniels for their advice throughout this work. We also want to thank Mr J. M. Theate and Mrs C. Marchand-Coquay for their valuable technical assistance.     

Ionophoric activity of the antibiotic X.14547 A in the mitochondrial membrane

Summary Release of Ca⁺⁺, Mg⁺⁺ and K⁺ by the carboxylic ionophore X-14547 A was studied in the mitochondrial membrane. A comparison was made with A.23187 (Calcimycin) and X.537 A (Lasalocid A) under the same experimental conditions. It was shown that in this test system X.14547 A is primarily a K⁺ carrier comparable with X.537 A.     

A threshold of transmembrane potential is required for mitochondrial dynamic balance mediated by DRP1 and OMA1

As an organellar network, mitochondria dynamically regulate their organization via opposing fusion and fission pathways to maintain bioenergetic homeostasis and contribute to key cellular pathways. This dynamic balance is directly linked to bioenergetic function: loss of transmembrane potential across the inner membrane (Δψ _m) disrupts mitochondrial fission/fusion balance, causing fragmentation of the network. However, the level of Δψ _m required for mitochondrial dynamic balance, as well as the relative contributions of fission and fusion pathways, have remained unclear. To explore this, mitochondrial morphology and Δψ _m were examined via confocal imaging and tetramethyl rhodamine ester (TMRE) flow cytometry, respectively, in cultured 143B osteosarcoma cells. When normalized to the TMRE value of untreated 143B cells as 100%, both genetic (mtDNA-depleted ρ⁰) and pharmacological [carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-treated] cell models below 34% TMRE fluorescence were unable to maintain mitochondrial interconnection, correlating with loss of fusion-active long OPA1 isoforms (L-OPA1). Mechanistically, this threshold is maintained by mechanistic coordination of DRP1-mediated fission and OPA1-mediated fusion: cells lacking either DRP1 or the OMA1 metalloprotease were insensitive to loss of Δψ _m, instead maintaining an obligately fused morphology. Collectively, these findings demonstrate a mitochondrial ‘tipping point’ threshold mediated by the interaction of Δψ _m with both DRP1 and OMA1; moreover, DRP1 appears to be required for effective OPA1 maintenance and processing, consistent with growing evidence for direct interaction of fission and fusion pathways. These results suggest that Δψ _m below threshold coordinately activates both DRP1-mediated fission and OMA1 cleavage of OPA1, collapsing mitochondrial dynamic balance, with major implications for a range of signaling pathways and cellular life/death events.     

The effect of temperature on Ca2+ uptake and Ca2+-activated ATP hydrolysis by cardiac sarcoplasmic reticulum

Zusammenfassung Der Ca²⁺-Transport und die Ca²⁺-aktivierte ATP Hydrolyse (ATP extra Spaltung) durch Membranen des cardialen sarkoplasmatischen Retikulums zeigen die gleiche Temperaturabhängigkeit. Die Aktivierungsenergie der Ca²⁺-Aufnahme und der ATP extra Spaltung, gemessen bei Anwesenheit von Oxalat, beträgt 16.65±0.87 und 17.93±0.49 Kcal/Mol^–1.     

Patient participation in the clinical encounter and clinical practice guidelines: The case of patients’ participation in a GRADEd world

It is widely acknowledged that the patient's perspective should be considered when making decisions about how her care will be managed. Patient participation in the decision making process may play an important role in bringing to light and incorporating her perspective. The GRADE framework is touted as an evidence-based process for determining recommendations for clinical practice; i.e. determining how care ought to be managed. GRADE recommendations are categorized as “strong” or “weak” based on several factors, including the “values and preferences” of a “typical” patient. The strength of the recommendation also provides instruction to the clinician about when and how patients should participate in the clinical encounter, and thus whether an individual patient's values and preferences will be heard in her clinical encounter. That is, a “strong” recommendation encourages “paternalism” and a “weak” recommendation encourages shared decision making. We argue that adoption of the GRADE framework is problematic to patient participation and may result in care that is not respectful of the individual patient's values and preferences. We argue that the root of the problem is the conception of “values and preferences” in GRADE – the framework favours population thinking (e.g. “typical” patient “values and preferences”), despite the fact that “values and preferences” are individual in the sense that they are deeply personal. We also show that tying the strength of a recommendation to a model of decision making (paternalism or shared decision making) constrains patient participation and is not justified (theoretically and/or empirically) in the GRADE literature.