Physical performance level in sarcomeric mitochondria creatine kinase knockout mouse model throughout ageing.

PURPOSE: The objective of the present study was to establish the role of sarcomeric mitochondrial creatine kinase (Mt-CK) in muscle energy output during exercise in a murine model of ageing (the Mt-CK knock-out mouse, Mt-CK-/-). METHODS: Three age groups of Mt-CK-/- mice and control male mice (6, 9, and 18 months of age) underwent incremental treadmill running tests. The maximum speed (Vpeak) and maximal oxygen consumption (VO2peak) values were recorded. Urine samples were analyzed using metabolomic techniques. The skeletal muscle (quadriceps) expression of proteins involved in mitochondria biogenesis, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and dynamin-related GTPase mitofusin 2 (Mnf2) were quantified. RESULTS: The VO2 peak (normalized to heart weight: HW) of 18-month-old (mo) Mt-CK-/- mice was 27% (p < 0.001) lower than in 18-mo control mice. The VO2peak/HW ratio was 29% (p < 0.001) lower in 18-mo Mt-CK-/- mice than in 6-mo (p < 0.001) and 32% (p < 0.001) than 9-mo Mt-CK-/- mice. With a 0° slope, Vpeak was 10% (p < 0.05) lower in 18-mo Mt-CK-/- mice than in 6-mo Mt-CK-/- mice but did not differ when comparing the 18-mo and 6-mo control groups. The skeletal muscles weight normalized on body weight in 6-mo Mt-CK-/- were 13 to 14% (p < 0.001, p < 0.05) lower versus the 6-mo control, in addition, the presence of branched-chain amino acids in the urine of 6-mo Mt-CK-/- mice suggests an imbalance in protein turnover (catabolism rather than anabolism) but we did not observe any age-related differences. The expression of PGC-1α and Mnf2 proteins in the quadriceps showed that age-related effects were more prominent than genotype effects. CONCLUSION: The present study showed ageing is potentialized by Mt-CK deficiency with regard to VO2peak, Vpeak and mitochondrial protein expression. Our results support that Mt-CK-/- mice undergo physiological adaptations, enabling them to survive and to perform as well as wild-type mice. Furthermore, it is possible that these adaptations in Mt-CK-/- mice have a high energy cost and might trigger premature ageing.

A new model of short acceleration-based training improves exercise performance in old mice.

In order to identify a more appealing exercise strategy for the elderly, we studied a mouse model to determine whether a less time-consuming training program would improve exercise performance, enzyme activities, mitochondrial respiration, and metabolomic parameters. We compared the effects of short-session (acceleration-based) training with those of long-session endurance training in 23-month-old mice. The short-session training consisted of five acceleration-based treadmill running sessions over 2 weeks (the acceleration group), whereas the endurance training consisted of five-one-hour treadmill sessions per week for 4 weeks (the endurance group). A control group of mice was also studied. In the acceleration group, the post-training maximum running speed and time to exhaustion were significantly improved, relative to pretraining values (+8% for speed, P<.05; +10% for time to exhaustion, P<.01). The post-training maximum running speed was higher in the acceleration group than in the endurance group (by 23%; P<.001) and in the control group (by 15%; P<.05). In skeletal muscle samples, the enzymatic activities of citrate synthase, lactate dehydrogenase, and creatine kinase were significantly higher in the acceleration group than in the endurance group. Furthermore, mitochondrial respiratory activity in the gastrocnemius was higher in the acceleration group than in the control group. A metabolomic urine analysis revealed a higher mean taurine concentration and a lower mean branched amino acid concentration in the acceleration group. In old mice, acceleration-based training appears to be an efficient way of increasing performance by improving both aerobic and anaerobic metabolism, and possibly by enhancing antioxidant defenses and maintaining muscle protein balance.

Pyruvate carboxylase deficiency: An underestimated cause of lactic acidosis.

Pyruvate carboxylase (PC) is a biotin-containing mitochondrial enzyme that catalyzes the conversion of pyruvate to oxaloacetate, thereby being involved in gluconeogenesis and in energy production through replenishment of the tricarboxylic acid (TCA) cycle with oxaloacetate. PC deficiency is a very rare metabolic disorder. We report on a new patient affected by the moderate form (the American type A). Diagnosis was nearly fortuitous, resulting from the revision of an initial diagnosis of mitochondrial complex IV (C IV) defect. The patient presented with severe lactic acidosis and pronounced ketonuria, associated with lethargy at age 23 months. Intellectual disability was noted at this time. Amino acids in plasma and organic acids in urine did not show patterns of interest for the diagnostic work-up. In skin fibroblasts PC showed no detectable activity whereas biotinidase activity was normal. We had previously reported another patient with the severe form of PC deficiency and we show that she also had secondary C IV deficiency in fibroblasts. Different anaplerotic treatments in vivo and in vitro were tested using fibroblasts of both patients with 2 different types of PC deficiency, type A (patient 1) and type B (patient 2). Neither clinical nor biological effects in vivo and in vitro were observed using citrate, aspartate, oxoglutarate and bezafibrate. In conclusion, this case report suggests that the moderate form of PC deficiency may be underdiagnosed and illustrates the challenges raised by energetic disorders in terms of diagnostic work-up and therapeutical strategy even in a moderate form.

Metabolomic profiling with NMR discriminates between biphosphonate and doxorubicin effects on B16 melanoma cells.

The metabolomic profiles of B16 melanoma cells were investigated in vitro with high resolution-magic angle spinning proton magnetic resonance spectroscopy and OPLS multivariate statistical analyse. We compared the profiles for untreated melanoma B16-F10 cells and Ca(2+) chelating EGTA, doxorubicin or BP7033 bisphosphonate treated cells. The two last molecules are known to induce anti-proliferative effects by different mechanisms of action in cells. Untreated and EGTA treated cells had similar profiles and were considered together as control cells. Several spectral regions could discriminate control from doxorubicin as well as BP7033 treated cells. Doxorubicin and BP7033 displayed distinct metabolic profiles. Important changes in neutral lipids and inositol were related to doxorubicin activity whereas BP7033 affected essentially phospholipids and alanine/lactate metabolism. These results provide new putative targets for both drugs. Metabolomics by NMR is shown here to be a good tool for the investigation of the mechanisms of action of drugs in pre-clinical studies.

Mechanism of interaction of sitamaquine with Leishmania donovani.

OBJECTIVES: This study focuses on the mechanism of interaction of sitamaquine with Leishmania donovani membranes, and its accumulation within the parasites. METHODS: A biomimetic model of the outer layer of a Leishmania plasma membrane was used to examine the interactions of sitamaquine with lipids. The plasma membranes of L. donovani promastigotes were depleted of sterol using cholesterol oxidase, in order to assess the importance of sterols in drug-membrane interactions. Sterols were quantified and sitamaquine susceptibility was assessed using the MTT test. Kinetics of sitamaquine accumulation and efflux were measured under different conditions. RESULTS: Sitamaquine interacts first with phospholipid anionic polar head groups and then with phospholipid acyl chains to insert within biological membranes and accumulates rapidly in the Leishmania cytosol according to a sterol-independent process. The rapid sitamaquine efflux observed was related to an energy-dependent mechanism since the intracellular amount of sitamaquine was enhanced three times in the absence of glucose and the efflux was inhibited in energy-depleted conditions. (1)H NMR analysis of motile lipid showed that sitamaquine did not affect lipid trafficking in Leishmania. CONCLUSIONS: We propose that sitamaquine rapidly accumulates in Leishmania by diffusion along an electrical gradient and is concentrated in the cytosol by an energy- and sterol-independent process. The affinity of sitamaquine for membranes was transitory and an energy-dependent efflux was demonstrated, suggesting the presence of an as yet uncharacterized transporter.

Membrane sterol depletion impairs miltefosine action in wild-type and miltefosine-resistant Leishmania donovani promastigotes.

OBJECTIVES: This study focuses on the importance of sterols in the action of miltefosine (hexadecylphosphocholine, HePC) against Leishmania donovani. METHODS: Plasma membranes of L. donovani promastigotes were depleted of sterol using methyl-beta-cyclodextrin (MCD) and cholesterol oxidase (CH-OX). Sterols were quantified and HePC susceptibility was assessed using the MTT test. A biomimetic model of the outer leaflet of a Leishmania plasma membrane was used to decipher the HePC-lipid interactions. RESULTS: CH-OX, which is known to act more specifically on condensed membranes, therefore at the level of lipid rafts, gave a better extraction yield in HePC-resistant parasites, confirming the more rigid structure of their membranes than those of wild-type parasites. Sterol depletion was responsible for a 40% decrease in HePC susceptibility in both wild-type and HePC-resistant parasites. Sterol repletion of the sterol-depleted parasites restored HePC susceptibility. The biomimetic model of the outer leaflet of a Leishmania plasma membrane confirmed that condensed microdomains were able to incorporate higher quantities of HePC than fluid ones and this result was amplified when the sterol concentration was increased. CONCLUSIONS: Sterol and lipid rafts probably play a significant role as an HePC reservoir providing a constant supply to the previously described transporter. In addition, (1)H NMR experiments suggested that HePC stimulated lipid trafficking in parasites.

N-acetyl functions and acetate detected by nuclear magnetic resonance spectroscopy of urine to detect renal dysfunction following aminoglycoside and/or glycopeptide antibiotic therapy.

BACKGROUND/AIMS: N-acetylneuraminidine (NeuNAc), N-acetylglutamine (GIcNAc) and acetate are metabolites present in normal urine. In patients treated with aminoglycosides and/or glycopeptides, elevation of these metabolites in urine suggests renal tubular injury. NeuNAc, GIcNAc and acetate are easily detected by magnetic resonance spectroscopy (MRS), in contrast to other bioanalytical methods. In the present study, these urinary metabolites were detected using MRS and compared with standard biochemical markers of renal injury in intensive care unit patients treated with aminoglycosides and/or glycopeptides. METHODS: 16 patients with clinical and biochemical signs of renal dysfunction were included in the study. Proton magnetic resonance spectra were obtained from 134 urine samples. The resonance intensity of NeuNAc, GIcNAc and acetate were reported relative to the resonance intensity of creatinine (ct). These ratios were compared with classical parameters of renal dysfunction, such as plasma creatinine and urea concentration, and 24-hour urine volume, by logistic regression and general linear models. RESULTS: Statistical analysis showed that changes in plasma creatinine and urea concentration were reliably reflected in changes in the NeuNAc/ct ratio, and that plasma urea concentration changes also correlated with the acetate/ct ratio; however, the GIcNAc/ct ratio was not related to these measures of overall renal function. CONCLUSIONS: NeuNAc/ct may be a useful marker of renal dysfunction in patients treated with aminoglycosides and glycopeptides; by MRS it can be both straightforward and informative to follow the renal function of patients treated with these antibiotics.

Solution conformation of an RNA--DNA hybrid duplex containing a pyrimidine RNA strand and a purine DNA strand.

RNA--DNA hybrid duplexes are involved in transcription, replication and reverse transcription of nucleic acids. Information on such duplexes may shed some light on the mechanism of these processes. For this purpose, the influence of base composition on the structure of a polypyrimidine--polypurine RNA--DNA duplex r(cucuccuucucuu). d(GAGAGGAAGAGAA) has been studied using 1H, 31P and 13C NMR experiments, molecular modeling (JUMNA program) and NOE back-calculation methods. The resulting structure of the 13-mer hybrid duplex shows that the RNA strand is in the expected A-type conformation while the DNA strand is in a very flexible conformation. In the DNA strand, the desoxyribose sugars retain the C2'-endo B-type conformation. The duplex helical parameters (such as inclination, twist and displacement of the bases) are close to the A-type conformation. No bending was observed for the global axis curvature. The major groove width is close to the B-form value and the minor groove width is intermediate between standard values for A and B-forms. These results are in favour of the independence of minor groove size (where RNase H interacts) and the base composition of the hybrid duplexes.

Differentiation of the P-gp and MRP1 multidrug resistance systems by mobile lipid 1H-NMR spectroscopy and phosphatidylserine externalization.

We have previously demonstrated that proton NMR spectra of fatty acid chains in erythroleukemia K562 wild-type cells and their MDR1 counterparts show variations related to the phenotype over-expressing the P-glycoprotein (P-gp). Human lung cancer cells whose multidrug resistance (MDR) counterparts over-express the multidrug resistance-associated protein MRP1 have not yet been studied by NMR. Both P-gp and MRP1 belong to the same ATP-binding cassette transporter superfamily. A comparison of NMR spectra from both these multidrug-resistance phenotypes showed that the results previously obtained on the MDR1 family are not valid for MRP1. Furthermore, flow cytofluorimetry studies with external phosphatidylserine labelling showed that P-gp and MRP1 overexpressions have strong but differentiated effects on cell lipid pools.

Magnetic resonance spectroscopy of cellular lipid extracts from sensitive, resistant and reverting K562 cells and flow cytometry for investigating the P-glycoprotein function in resistance reversion.

The proton NMR spectra of K562 cells contain resonances of lipids. When these cells acquire multidrug resistance phenotype, the NMR lipid signals are modified and partially recovered when the resistance is reversed. The goals of the present study are to elucidate the mechanism of the resistance phenotype reversion and to investigate the possible origin of lipid signals detected in whole cells with proton NMR spectroscopy. Therefore, the K562 drug-sensitive cell line, its adriamycin resistant counterpart and two reverting derivates, obtained by verapamil treatment and long term culture in drug-free medium, were used in this study. The P-glycoprotein (P-gp) pump function was measured by flow cytometry and lipids were extracted to be analysed by proton and phosphorus spectroscopy. The phenotype reversion is due to the decrease of the P-gp function and an increased entrance of anthracycline drug when compared with the resistant cells. The spectra obtained on extracts showed no modification of the fatty acid composition and of the ratio of total cholesterol to fatty acid content. A different phospholipid composition in sensitive and resistant cells was found, but the reversion of resistance did not produce a recovery of these lipids. Thus, the lipid NMR spectra of extracts could not explain the spectral modifications observed on whole cells, in relation to acquiring and reverting drug resistance. These results are in favour of a different lipid organization or of localization within the cell.

Current Awareness.

In order to keep subscribers up-to-date with the latest developments in their field, John Wiley & Sons are providing a current awareness service in each issue of the journal. The bibliography contains newly published material in the field of NMR in biomedicine. Each bibliography is divided into 9 sections: 1 Books, Reviews ' Symposia; 2 General; 3 Technology; 4 Brain and Nerves; 5 Neuropathology; 6 Cancer; 7 Cardiac, Vascular and Respiratory Systems; 8 Liver, Kidney and Other Organs; 9 Muscle and Orthopaedic. Within each section, articles are listed in alphabetical order with respect to author. If, in the preceding period, no publications are located relevant to any one of these headings, that section will be omitted.

Magnetic resonance spectroscopy and fluorescence microscopy to investigate mobile lipids in sensitive, resistant and reverting K562 cells and their membranes.

The erythroleukaemic K562 cell line and its adriamycin resistant counterpart were used to study resistance, its reversion and their consequences on the levels and localisation of lipids detected in proton nuclear magnetic resonance (NMR) spectra. On whole cells, the mobile lipids giving rise to a NMR signal were significantly decreased in the resistant cells when compared to the sensitive ones; these lipids recovered partially in the reverting cells. According to the spinlattice relaxation times (T1), the lipids detected appeared to be in a similar environment in sensitive and reverting cells. In membrane-enriched fractions, mobile lipid levels were not significantly different in the sensitive and reverting cell lines but decreased in resistant ones. Moreover, lipid droplets stained with a fluorescent Nile red lipophilic probe showed the presence of highly fluorescent particles in the samples in which NMR detected high levels of mobile lipids. These results suggest the participation of cytosolic lipid droplets in NMR signals in drug sensitive and reverting cells and open the question of the relative roles of these droplets and of the membrane lipids in the lipid metabolic pathways associated with drug resistance in cancer cells.

Trimetazidine prevents renal injury in the isolated perfused pig kidney exposed to prolonged cold ischemia.

BACKGROUND: Ischemia caused by cold storage (CS) and reperfusion of the kidney is often responsible for delayed graft function after transplantation. Significant attention has been focused on the cascade of events involved in ischemia-reperfusion injury, with the objective of identifying drugs to ameliorate the functional damage that occurs. METHODS: The purpose of this study was to evaluate the renal function of isolated perfused pig kidneys after 48 hr of CS with Euro-Collins (EC) solution plus trimetazidine (EC+TMZ), standard EC solution, or University of Wisconsin (UW) solution. Normothermic isolated perfused pig kidneys were randomized into five experimental groups: (A) control group (cold flush with cold heparinized saline and immediately reperfused; n=6); (B) cold flush with cold heparinized saline with TMZ (10(-6) M), n=6; (C) 48 hr of CS with EC and reperfusion (n=8); (D) 48 hr of CS with EC+TMZ alone and reperfusion (n=8); (E) 48 hr of CS with UW and reperfusion (n=8). Proton nuclear magnetic resonance spectroscopy and biochemical studies were performed for the functional evaluation during reperfusion. Lipid peroxidation was also determined. Histological examination (optical and electron microscopy) was performed after CS and reperfusion. RESULTS: Using TMZ, the renal perfusate flow rate as well as the glomerular filtration rate and proximal tubular function were significantly improved. This improvement of renal function during reperfusion was correlated with a less significant cellular and interstitial edema. In addition, tubular injury markers were significantly lower in the group preserved with EC+TMZ, and TMZ reduced lipid peroxidation dramatically during reperfusion. CONCLUSIONS: The addition of TMZ to the EC solution increased the preservation quality and renal tubular function, and gave protection from reperfusion injury better than EC alone or UW. These results strongly suggest that TMZ has a cytoprotective effect and may therefore be useful for kidney preservation.

Lipid signals detected by NMR proton spectroscopy of whole cells are not correlated to lipid droplets evidenced by the Nile red staining.

Nile red staining was used to detect lipid droplets in the K562 cell line sensitive and resistant to adriamycin and their resistance-reversing counterparts. The staining obtained was compared to the intensity of lipid signal detected in proton nuclear magnetic resonance spectra. From the four cell lines used, a lack of correlation was observed between the NMR signal and the Nile red staining. For example, the sensitive K562 cells, with the highest level of NMR signals, showed only few cells containing lipid droplets. We concluded that is lipid droplets can participate to lipid signal in NMR spectra, other lipids must also participate to these resonance.

Evaluation of normothermic ischemia and simple cold preservation injury in pig kidney by proton nuclear magnetic resonance spectroscopy.

The isolated perfused pig kidney (IPK) model was used to mimic the non-heart-beating donor situation. This model was performed to assess initial renal functions after normothermic ischemia, cold flush, and 24 hr cold-storage preservation (CSP) with Euro-Collins and to determine normothermic ischemia and reperfusion impairment by biochemical, histological, and proton nuclear magnetic resonance (NMR) spectroscopy analysis. Twenty-four pig kidneys were used. There were three experimental groups: Group 1 (G1), control kidneys flushed with cold heparinized saline and immediately perfused; Group 2 (G2), cold flush followed by 24 hr CSP and reperfusion; and Group 3 (G3), 30 min of normothermic ischemia followed by cold flush and 24 hr CSP and reperfusion. Kidneys were perfused for 2 hr at 37.5 degrees C for functional evaluation. Perfusate flow rate is significantly different for G3 (P < 0.01). Glomerular filtration rate is less in G3 (P < 0.05). Fractional reabsorptions of sodium (FRNa+) and glucose (Glc) excretion in urine are different in G3 (P < 0.01 and P < 0.05, respectively). Amino acid excretion in NMR spectroscopy was higher in G3 (P < 0.05). Elevated levels of trimethylamine-N-oxide (TMAO) and lactate (Lac) were detected by proton NMR spectroscopy in G2 and particularly G3. A peak is present in G3 and related with poor glomerular and tubular functions and worse histological data. Malondialdehyde tissue level was higher in G3. This study shows that the IPK with proton NMR spectroscopy may be a useful method in the evaluation of kidneys after cold ischemia and transplantation. This model might be suitable for a variety of experimental protocols, particularly to improve functional performance after ischemia and reperfusion.