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Background We measured the functional and metabolic status of hearts submitted to normothermic ischemia before preservation through the use of an ex vivo pig heart model to assess the feasibility of donation after cardiac death (DCD) in heart transplantation. Methods Ten pigs were separated into 2 groups: control ( n = 6, brain-dead group) and DCD ( n = 4, heart donation after cardiac death). In the control group, hearts were excised 20 minutes after the brachiocephalic trunk cross-clamping and were immediately reperfused. In DCD, hearts were excised 20 minutes after exsanguination and asphyxia, stored in the Centre de Resonance Magnetique Biologique et Medicale (CRMBM) solution for 2 hours, and then were reperfused. Cardioplegic arrest was induced with the use of 1 L of CRMBM solution (4°C) and the heart was reperfused for 60 minutes through the use of an ex vivo perfusion system in Langendorff mode with normothermic autologous blood. During reperfusion, functional parameters were analyzed. Biochemical assays were performed in myocardial effluents and freeze-clamped hearts. Results No electromechanical activity was found in DCD compared with control. Creatine kinase (CK) was higher at 2 minutes of reperfusion in DCD versus control ( P = .005). Adenosine triphosphate was lower in DCD versus control ( P = .0019). Malondialdehyde, an oxidative stress index, was present only in DCD. The nitric oxide (NO) pathway was impaired in DCD versus control, with lower eNOS expression ( P P = .04). Conclusions We reported no cardiac functional and metabolic recovery in the DCD group after normothermic ischemia and reperfusion, which indicates that a single immersion of the cardiac graft during storage does not provide an optimal protection. New strategies in heart preservation are necessary for recruiting heart donation after cardiac death.

While muscle damage resulting from electrically-induced muscle isometric contractions has been reported in humans, animal studies have failed to illustrate similar deleterious effects and it remains to be determined whether these conflicting results are related to differences regarding experimental procedures or to species. We have investigated in vivo , in rat gastrocnemius muscles, using experimental conditions as close as possible to those used in humans (i.e., muscle length, number of contractions, stimulated muscle), the effects of a single bout of neuromuscular electrical stimulation (NMES). Maximal tetanic force was measured before, immediately after and 1h and 1, 2, 3, 7 and 14days after NMES. Magnetic resonance imaging measurements, including volume of gastrocnemius muscles and proton transverse relaxation time ( T 2 ) were performed at baseline and 3, 7, and 14days after the NMES session. Control animals did not perform any exercise and measurements were recorded at the same time points. For both groups, blood creatine kinase (CK) activity was measured within the first 3days that followed the initial evaluation. Maximal tetanic force decreased immediately after NMES whereas measurements performed 1h and the days afterwards were similar to the baseline values. CK activity, muscle volume and T 2 values were similar throughout the experimental protocol between the two groups. Under carefully controlled experimental conditions, isometric NMES per se did not induce muscle damage in rat gastrocnemius muscles on the contrary to what has been repeatedly reported in humans. Further experiments would then be warranted in order to clearly delineate these differences and to better understand the physiological events associated with muscle damage resulting from NMES-induced isometric contractions.

Development of surrogate markers is necessary to assess the potential efficacy of new therapeutics in Niemann–Pick Disease Type C (NP-C). In the present study, magnetization transfer ratio (MTR) imaging, a quantitative MRI imaging technique sensitive to subtle brain microstructural changes, was applied in two patients suffering from adult NP-C. Statistical mapping analysis was performed to compare each patient's MTR maps with those of a group of 34 healthy controls to quantify and localize the extent of brain injury of each patient. Using this method, pathological changes were evidenced in the cerebellum, the thalami and the lenticular nuclei in both patients and also in the fronto-temporal cortices in the patient with the worse functional deficit. In addition, white matter changes were located in the midbrain, the cerebellum and the fronto-temporal lobes in the patient with the higher level of disability and in only one limited periventricular white matter region in the other patient. A 6-month follow-up was performed in the patient with the lower functional deficit and evidenced significant extension of grey matter (GM) and white matter (WM) injuries during the following period (14% of increased injury for GM and 53% for WM). This study demonstrates that significant brain injury related to clinical deficit can be assessed in vivo in adult NP-C using MTR imaging. Although preliminary, these findings suggest that MTR imaging may be a relevant candidate for the development of biomarker in NP-C.

PASTA (penicillin-binding protein and serine/threonine kinase associated) modules are found in penicillin-binding proteins and bacterial serine/threonine kinases mainly from Gram-positive Firmicutes and Actinobacteria. They may act as extracellular sensors by binding peptidoglycan fragments. We report here the first crystal structure of a multiple-PASTA domain from Ser/Thr kinase, that of the protein serine/threonine kinase 1 (Stk1) from the Firmicute Staphylococcus aureus. The extended conformation of the three PASTA subunits differs strongly from the compact conformation observed in the two-PASTA domain of penicillin-binding protein PBP2x, whereas linear conformations were also reported for two-subunit fragments of the four-PASTA domain of the Actinobacteria Mycobacterium tuberculosis studied by liquid NMR. Thus, a stretched organization appears to be the signature of modular PASTA domains in Ser/Thr kinases. Signal transduction to the kinase domain is supposed to occur via dimerization and ligand binding. A conserved X-shaped crystallographic dimer stabilized by intermolecular interactions between the second PASTA subunits of each monomer is observed in the two crystal forms of Stk1 that we managed to crystallize. Extracellular PASTA domains are composed of at least two subunits, and this molecular assembly is a plausible candidate for the biological dimer. We have also performed docking experiments, which predict that the hinge regions of the PASTA domain can accommodate peptidoglycan. Finally, a three-dimensional homology molecular model of full-length Stk1 was generated, suggesting an interaction between the kinase domain and the cytoplasmic face of the plasma membrane via a eukaryotic-like juxtamembrane domain. A comprehensive activation mechanism for bacterial Ser/Thr kinases is proposed with the support of these structural data.

Although citrulline malate (CM; CAS 54940-97-5, Stimol) is used against fatigue states, its anti-asthenic effect remains poorly documented. The objective of this double-blind study was to evaluate the effect of oral ingestion of CM on a rat model of asthenia, using in situ (31)Phosphorus magnetic resonance spectroscopy ((31)P-MRS). Muscle weakness was induced by intraperitoneal injections of Klebsiella pneumoniae endotoxin (lipopolysaccharides at 3 mg/kg) at t(0) and t(0)+24 h. For each animal, muscle function was investigated strictly non-invasively before (t(0)-24 h) and during (t(0)+48 h) endotoxemia, through a standardized rest-stimulation-recovery protocol. The transcutaneous electrical stimulation protocol consisted of 5.7 min of repeated isometric contractions at a frequency of 3.3 Hz, and force production was measured with an ergometer. CM supplementation in endotoxemic animals prevented the basal phosphocreatine/ATP ratio reduction and normalized the intracellular pH (pH(i)) time-course during muscular activity as a sign of an effect at the muscle energetics level. In addition, CM treatment avoided the endotoxemia-induced decline in developed force. These results demonstrate the efficiency of CM for limiting skeletal muscle dysfunction in rats treated with bacterial endotoxin.

Bacterial tyrosine-kinases have been demonstrated to participate in the regulation of capsule polysaccharides (CPS) and exopolysaccharides (EPS) production and export. However, discrepant data have been reported on the molecular mechanism responsible for this regulation depending on the bacterial species analyzed. Special attention was previously paid to the tyrosine-kinase Wzcca of Escherichia coli K-12, which is involved in the production of the exopolysaccharide, colanic acid, and autophosphorylates by using a cooperative two-step process. In this work, we took advantage of these observations to investigate in further detail the effect of Wzcca phosphorylation on the colanic acid production. First, it is shown that expression of the phosphorylated form of Wzc prevents production of colanic acid whereas expression of the non-phosphorylated form allows biosynthesis of this exopolysaccharide. However, we provide evidence that, in the latter case, the size distribution of the colanic acid polymer is less scattered than in the case of the wild-type strain expressing both phosphorylated and non-phosphorylated forms of Wzc. It is then demonstrated that colanic acid production is not merely regulated by an on/off mechanism and that, instead, both phosphorylated and non-phosphorylated forms of Wzc are required to promote colanic acid synthesis. Moreover, a series of data suggests that besides the involvement of phosphorylated and non-phosphorylated forms of Wzc in the production of colanic acid, two particular regions of this kinase play as such an important role in the synthesis of this exopolysaccharide: a proline-rich domain located in the N-terminal part of Wzcca, and a tyrosine cluster present in the C-terminal portion of the enzyme. Furthermore, considering that polysaccharides are known to facilitate bacterial resistance to certain environmental stresses, it is shown that the resistance of E. coli to desiccation is directly connected with the phosphorylation state of Wzcca.

International audience; The aim of this study was to better understand the significance of interictal changes in water molecule diffusivity defined by diffusion-weighted imaging (DWI) in frontal lobe epilepsy (FLE), as well as to test the accuracy of interictal DWI in the definition of the epileptogenic zone (EZ).DWI was carried out in 14 patients with refractory FLE (9 negative-MRI) as well as in 25 controls. Statistical mapping analysis (SPM2) of diffusivity maps was used to detect, for each subject, significant diffusivity alterations. We then studied the relationships between diffusion and depth recorded electrical abnormalities. Clinical correlates of the extent of diffusivity changes were also tested. We found areas of significantly increased diffusivity (SID) in 13 patients. Eight had SID in the EZ, 9 within the irritative zone (IZ) and 12 outside, mainly in connected areas. We found a correlation between the extent of SID and the duration of epilepsy (p corrected = 0.026, R = 0.621). In addition, SID was significantly less widespread in negative-MRI patients (p = 0.028). However, we found no significant differences concerning either seizure frequency (p = 0.302), seizure generalization (p = 0.841), history of status (p = 0.396), or surgical outcome (p = 0.606). We suggest that SID in normal appearing areas is not a specific signature of epileptogenicity in FLE, and is more likely to reflect multifactorial and potentially evolving neuro-glial injuries.

International audience; The role of protein-tyrosine kinases in bacterial polymyxin resistance was assessed by both genetic and biochemical approaches. Each of the two genes, wzc and etk, encoding protein-tyrosine kinases in Escherichia coli, was knocked out by using the PCR-based method of one-step inactivation of chromosomal genes, and the corresponding mutant strain was assayed in each case for resistance to different concentrations of polymyxin B by measuring the percentage of surviving cells. The resistance of a double knock-out wzc-etk-mutant was also analyzed and complementation experiments were performed by checking the effect of plasmid vectors expressing either Wzc or Etk. Our results concurred in showing that protein-kinase Wzc is not essential for polymyxin resistance, whereas protein-kinase Etk appears to play a key role in such antibiotic resistance. This newly found specific function of Etk reinforces the concept that protein-tyrosine kinases are involved in distinct facets of bacterial physiology.The role of protein-tyrosine kinases in bacterial polymyxin resistance was assessed by both genetic and biochemical approaches. Each of the two genes, wzc and etk, encoding protein-tyrosine kinases in Escherichia coli, was knocked out by using the PCR-based method of one-step inactivation of chromosomal genes, and the corresponding mutant strain was assayed in each case for resistance to different concentrations of polymyxin B by measuring the percentage of surviving cells. The resistance of a double knock-out wzc-etk-mutant was also analyzed and complementation experiments were performed by checking the effect of plasmid vectors expressing either Wzc or Etk. Our results concurred in showing that protein-kinase Wzc is not essential for polymyxin resistance, whereas protein-kinase Etk appears to play a key role in such antibiotic resistance. This newly found specific function of Etk reinforces the concept that protein-tyrosine kinases are involved in distinct facets of bacterial physiology.

Following our previous reports based on parametric MRI methods (T 2 -weighted MRI, statistical mapping analysis of magnetization transfer ratio images and functional MRI) applied to a population of 18 patients with clinically isolated syndrome suggestive of multiple sclerosis, we have reviewed the possible structural and functional surrogates of MS that could explain the subtle cognitive impairment related to attention and working memory deficits evaluated with paced auditory serial addition test (PASAT). We propose that the brain substrates underlying cognitive impairment observed at the very early stage of MS are multifactorial. Several components could influence PASAT performances in patients: i) the extent of diffuse white matter damage, ii) the location of visible and non visible lesions, iii) the connectivity efficiency between distant brain functional areas involved in working memory processes and iv) the cortical reorganization. Nevertheless, individually, each of these parameters may have few influences on PASAT performance in patients. Using a multiregression model built with independent MR parameters, a very good evaluation of PASAT scores has been obtained in this limited number of patients explaining 90% of the variance. In conclusion, the different aspects of tissue and functional pathological brain underpinnings must be accounted to monitor accurately new therapeutic strategies for the treatment of early cognitive deficits related to MS.

During aerobic growth of Escherichia coli on acetate, the component parts of the 'acetate switch' are turned-on as a consequence of direct competition, on the one hand, between phosphotransacetylase (PTA) and alpha-ketoglutarate dehydrogenase (alpha-KGDH) for their common co-factor free-CoA (HS-CoA) and, on the other hand, between isocitrate lyase (ICL) and isocitrate dehydrogenase (ICDH) for their common substrate isocitrate. Flux analysis revealed that competitions at both junctions in central metabolism are resolved in a precise way, so that the fraction of HS-CoA flux processed through PTA for biosynthesis relative to that processed through alpha-KGDH for energy generation, matches that observed for isocitrate flux through ICL relative to ICDH at the junction of isocitrate. Whereas the mechanism involved in the partition of carbon flux at the level of HS-CoA in central metabolism remains to be unravelled, the competition at the junction of isocitrate is resolved by the reversible phosphorylation/inactivation of ICDH and the operation of the glyoxylate bypass, the expression of which is subject to regulation at the transcriptional and translational levels as well as being dependent on growth rate.During aerobic growth of Escherichia coli on acetate, the component parts of the 'acetate switch' are turned-on as a consequence of direct competition, on the one hand, between phosphotransacetylase (PTA) and alpha-ketoglutarate dehydrogenase (alpha-KGDH) for their common co-factor free-CoA (HS-CoA) and, on the other hand, between isocitrate lyase (ICL) and isocitrate dehydrogenase (ICDH) for their common substrate isocitrate. Flux analysis revealed that competitions at both junctions in central metabolism are resolved in a precise way, so that the fraction of HS-CoA flux processed through PTA for biosynthesis relative to that processed through alpha-KGDH for energy generation, matches that observed for isocitrate flux through ICL relative to ICDH at the junction of isocitrate. Whereas the mechanism involved in the partition of carbon flux at the level of HS-CoA in central metabolism remains to be unravelled, the competition at the junction of isocitrate is resolved by the reversible phosphorylation/inactivation of ICDH and the operation of the glyoxylate bypass, the expression of which is subject to regulation at the transcriptional and translational levels as well as being dependent on growth rate.

Following our previous reports based on parametric MRI methods (T(2)-weighted MRI, statistical mapping analysis of magnetization transfer ratio images and functional MRI) applied to a population of 18 patients with clinically isolated syndrome suggestive of multiple sclerosis, we have reviewed the possible structural and functional surrogates of MS that could explain the subtle cognitive impairment related to attention and working memory deficits evaluated with paced auditory serial addition test (PASAT). We propose that the brain substrates underlying cognitive impairment observed at the very early stage of MS are multifactorial. Several components could influence PASAT performances in patients: i) the extent of diffuse white matter damage, ii) the location of visible and non visible lesions, iii) the connectivity efficiency between distant brain functional areas involved in working memory processes and iv) the cortical reorganization. Nevertheless, individually, each of these parameters may have few influences on PASAT performance in patients. Using a multiregression model built with independent MR parameters, a very good evaluation of PASAT scores has been obtained in this limited number of patients explaining 90% of the variance. In conclusion, the different aspects of tissue and functional pathological brain underpinnings must be accounted to monitor accurately new therapeutic strategies for the treatment of early cognitive deficits related to MS.

Resume Les rhabdomyolyses suscitent de nos jours un regain d'interet. Qu'elles soient toxiques, medicamenteuses, infectieuses, associees a un effort, a un ecrasement musculaire ou a une maladie (dystrophinopathies, myopathies metaboliques) il est important de cerner le contexte physiopathologique pour circonscrire ce processus de lyse musculaire le plus rapidement possible et en eviter la recidive. Cette mise au point presente les mecanismes physiopathologiques, les differents contextes nosologiques et les modalites d'exploration des rhabdomyolyses avec un accent tout particulier sur les methodes d'exploration non invasive.

Rhabdomyolysis has sparked new interest in recent years. The causes of rhabdomyolysis include drugs and other toxic agents, infections, physical exertion, crush injury, and muscle diseases (dystrophinopathies and metabolic myopathies). Prompt identification of the pathophysiological mechanism is the key to rapid control of the acute episode and to prevention of recurrences. In this update, we discuss the pathophysiological mechanisms and nosology of rhabdomyolysis, as well as diagnostic investigations, with special emphasis on noninvasive methods.

Presurgical evaluation of frontal lobe epilepsy (FLE) remains a challenging issue and frequently requires invasive depth electrode recording. In this study, we aimed at evaluating the potential usefulness of a non-invasive technique such as proton magnetic resonance spectroscopic imaging ((1)H-MRSI) in the presurgical evaluation of FLE and at investigating the potential electrophysiological correlates of the metabolic disturbances as defined by (1)H-MRSI. We compared the distribution of (1)H-MRSI abnormalities with the electrophysiological abnormalities defined by stereo-electroencephalography (SEEG) recording in 12 patients presenting with several subtypes of FLE. We also used 12 control subjects in order to obtain normative (1)H-MRSI data. We used a multilevel (1)H-MRSI protocol to better sample the principal regions of the frontal lobe. We also applied a metabolic mapping technique allowing a visual display of metabolic data. A significant decrease of both N-acetyl-aspartate/phosphocreatine-creatine and N-acetyl-aspartate/(choline-compounds + phosphocreatine-creatine) ratios was observed in regions involved in the epileptogenic zone (EZ) and/or the irritative zone (IZ) compared to regions without electrical abnormalities in the same patients (P = 0.044 and P = 0.018, respectively), and also compared to controls (P = 0.004 and P = 0.0001, respectively). No significant differences in metabolic ratios were observed between those regions involved in the EZ and those involved in the IZ only. Our results suggest a link between the relative decrease of N-acetyl-aspartate and the EZ as well as the IZ in FLE. Thus, multilevel (1)H-MRSI protocol may add pertinent information during the non-invasive presurgical evaluation of FLE.

Muscle fatigue, which is defined as the decline in muscle performance during exercise, may occur at different sites along the pathway from the central nervous system through to the intramuscular contractile machinery. Historically, both impairment of neuromuscular transmission and peripheral alterations within the muscle have been proposed to be involved in the development of fatigue. However, according to the more recent studies, muscle energetics would have a key role in this process. Intramyoplasmic accumulation of inorganic phosphate (P(i)) and limitation in ATP availability are frequently proposed as the causative factors of fatigue development. Although attractive, these hypotheses have been elaborated on the basis of experimental results obtained in vitro and their physiological relevance has never been clearly demonstrated in vivo. In that context, non-invasive methods such as 31-phosphorus magnetic resonance spectroscopy ((31)P MRS) and electromyographic (EMG) recordings have been employed to understand both metabolic and electrical aspects of muscle fatigue under physiological condition. The main results of these studies are reviewed in the present paper.

Introduction. - We aimed to characterize the physical capacities of a population of professional cyclists using anthropometrical, cardiovascular, respiratory, ventilatory, and energetic measures. Synthesis of facts. - Eight professional cyclists participated in this study. They performed over 1 day, 5 exercises (progressive and constant-load test) on a cyclo-ergometer. Conclusions. - Physical and physiological results confirmed previous observations done on professional cyclists. The values of maximal power tolerated and time to exhaustion at this power are reproducible during the day thus demonstrated the excellent capability to recover of the professional cyclists.

Interindividual variations in skeletal muscle metabolism make comparative analyses difficult. In this study, we have addressed the issue of capturing the variability of metabolic performance observed during muscle exercise in humans by using an original method of normalization. Metabolic changes induced by various kinds of exercise were investigated using 31 P magnetic resonance spectroscopy (MRS) at 4.7 T in 65 normal subjects (23 women and 42 men) and 12 patients with biopsy-proven muscular disorders. Large variations in the extent of PCr breakdown and intracellular acidosis were recorded among subjects and exercise protocols. For all the data pooled, the amplitude of mechanical performance accounts for 50% of these variations. When scaled to the work output, variations of PCr consumption account for 65% of pH changes through a linear relationship. This linear relationship was substantially improved (90%) when both variables were scaled to the square of work output performed (P1 and P2). By capturing most of the initial interindividual variability (90%), P1 vs. P2 relationship represents an ideal standardization procedure, independent of any anthropometric measurements. This relationship also discloses a significant link between the extent of PCr breakdown and intracellular acidosis regardless of exercise protocol. Moreover, changes in the slope of the P1 vs. P2 regression curve, as measured in old subjects and in selected patients, directly reflect alterations of energy production in muscle.

Net phosphocreatine (PCr) resynthesis during muscle contraction is a paradoxical phenomenon because it occurs under conditions of high energy demand. The metabolic underpinnings of this phenomenon were analyzed non-invasively using 31 P-magnetic resonance spectroscopy in rat gastrocnemius muscle ( n =11) electrically stimulated (7.6 Hz, 6 min duration) in situ under ischemic and normoxic conditions. During ischemic stimulation, [PCr] initially fell to a steady state (9±5% of resting concentration) which was maintained for the last 5 min of stimulation, whereas isometric force production decreased to a non-measurable level beyond 3 min. Throughout normoxic stimulation, [PCr] and force production declined to a steady state after respectively 1 min (5±3% of resting concentration) and 3.25 min (21±8% of initial value) of stimulation. Contrary to the observations under ischemia, a paradoxical net PCr resynthesis was recorded during the last 2 min of normoxic stimulation and was not accompanied by any improvement in force production. These results demonstrate that the paradoxical net PCr resynthesis recorded in contracting muscle relies exclusively on oxidative energy production and could occur in inactivated fibers, similarly to PCr resynthesis during post-exercise recovery.

The regulation of transcription of the pckA gene coding for phosphoenolpyruvate carboxykinase in Escherichia coli was analysed by site-directed mutagenesis of the promoter region and measurement of in vitro transcription initiation. Mutation of the guanine residue at position –14 to either cytosine or thymine was found to result in a drastic decrease of transcription, even in the presence of the natural –35 hexamer TTTCCA that differs by only two nucleotides from the consensus sequence TTGACA. It was concluded that the promoter region of pckA contains an extended –10 module, 5`-TG-3`, located one base upstream of the –10 hexamer, which is crucial for transcription.

High resolution magnetic resonance spectroscopy (MRS) of cerebrospinal fluid (CSF) is a non-destructive analytical method which allows rapid and simultaneous detection of molecules involved in intermediary and oxidative metabolic pathways. We developed a protocol suitable for routine MRS analysis of lyophilized CSF samples. This procedure guarantees sample integrity, from CSF collection to spectrum acquisition. MRS analysis of blood serum was included in our protocol as a complementary method to CSF analysis. This protocol can contribute to establish MRS of CSF as a new analytical tool to better understand the metabolic processes involved in neurological diseases. Themes: Other systems of the CNS Topics: Brain metabolism and blood flow

This study examined the influence of metabolic substrates on the effects of trimetazidine on functional and metabolic aspects of the ischemic reperfused heart. Isovolumic rat hearts were submitted to a 30-minute period of global mild ischemia (coronary flow decreased by an average of 70%) and then reperfused at constant preischemic coronary flow rate. Either glucose (11 m M ) or glucose and palmitic acid (0.1 m M ) were used as metabolic substrates. Trimetazidine (6 × 10 −7 M ) markedly reduced the increase in diastolic pressure that occurred on reperfusion after the ischemic episode, whatever the exogenous substrate used. However, in those hearts that received fatty acid, the postischemic increase in diastolic pressure was abolished. Ischemia-induced increase in acyl carnitine levels—determined as indicators of fatty acid utilization by myocardial cells—was significantly decreased by trimetazidine in those hearts receiving fatty acid. Also, similar effects to those of trimetazidine on the postischemic increase in diastolic pressure and on tissue levels of acyl carnitine were obtained in the presence of dichloroacetate. Moreover, the presence of trimetazidine was associated with a reduction in the intracellular pH decrease during ischemia in those hearts receiving fatty acid. Combined with previous studies, these results suggest that an improved metabolic balance by trimetazidine may well consequently decrease the ionic imbalance after a transient period of ischemia.

Skeletal muscle energetics of seven hyperthyroid patients were investigated throughout a rest-exercise-recovery protocol using phosphorus-31 magnetic resonance spectroscopy (31P MRS) to quantitatively document in vivo the metabolic bases of impaired muscle performance in hyperthyroidism. The contributions of the main pathways of adenosine triphosphate (ATP) synthesis to energy production and proton efflux were measured and compared with results from normal muscle. At rest, a reduced concentration of phosphocreatine (PCr) was calculated for hyperthyroid patients when compared with controls, whereas pH and concentrations of inorganic phosphate (Pi) and phosphomonoesters (PME) were not different from controls. During exercise, the analysis of changes in pH and PCr concentration demonstrated that (1) at the onset of exercise, the magnitude of glycolysis activation is significantly larger for patients, resulting in a marked pH decrease; (2) the energy cost of exercise is higher for patients as compared with controls performing the same amount of work; and (3) both anaerobic and aerobic pathways are significantly more activated in the hyperthyroid group throughout the 3 minutes of exercise. During recovery, the rates of proton efflux and PCr resynthesis were similar in both groups, excluding any alteration in oxidative function and proton handling as a cause of initial glycolytic hyperactivation. The increased energy cost measured for patients during exercise evidences an increased need for energy, which is (1) probably linked to the existence of additional ATP-consuming mechanism(s), and (2) supported by hyperactivation of both aerobic and anaerobic pathways. These findings imply that, all things equal, a hyperthyroid muscle requires more energy to function than normal, and as a result is potentially more fatiguable.

The ptp gene of Acinetobacter johnsonii was previously reported to encode a low-molecular-mass protein, Ptp, whose amino acid sequence, predicted from the theoretical analysis of the nucleotide sequence of the gene, exhibits a high degree of similarity with those of different eukaryotic and prokaryotic phosphotyrosine-protein phophatases. We have now overexpressed the ptp gene in Escherichia coli cells, purified the Ptp protein to homogeneity by a single-step chromatographic procedure, and analysed its functional properties. We have shown that Ptp can catalyse the dephosphorylation of p-nitrophenyl phosphate and phosphotyrosine, but has no effect on phosphoserine or phosphothreonine. Its activity is blocked by ammonium molybdate and sodium orthovanadate, which are strong inhibitors of phosphotyrosine-protein phosphatases, as well as by N-ethylmaleimide and iodoacetic acid. Such specificity of Ptp for phosphotyrosine has been confirmed by the observation that it can dephosphorylate endogenous proteins phosphorylated on tyrosine, but not proteins modified on either serine or threonine. In addition, Ptp has been shown to quantitatively dephosphorylate two exogenous peptides, derived respectively from leech hirudin and human gastrin, previously phosphorylated on tyrosine. Moreover, site-directed mutagenesis experiments performed on Cys11 and Arg16, which are both present in the sequence motif (H/V)C(X5)R(S/T) typical of eukaryotic phosphotyrosine-protein phosphatases, have demonstrated that each amino acid residue is essential for the catalytic activity of Ptp. Taken together, these data provide evidence that Ptp is a member of the phosphotyrosine-protein phosphatase family. Furthermore, in search for the biological function of Ptp, we have found that it can specifically dephosphorylate an endogenous protein kinase, termed Ptk, which is known to autophosphorylate at multiple tyrosine residues in the inner membrane of Acinetobacter johnsonii cells. This represents the first identification of a protein substrate for a bacterial phosphotyrosine-protein phosphatase, and therefore constitutes a possible model for analysing the role of reversible phosphorylation on tyrosine in the regulation of microbial physiology.

La SRM cerebrale du proton detecte de maniere non invasive des pools de metabolites cellulaires. Dans les abces bacteriens, elle permet de realiser le diagnostic differentiel d'avec les tumeurs gliales malignes. Dans le sida, la SRM detecte, avant l'IRM, la presence d'une encephalopathie liee au VIH et offre une evaluation quantifiee de la reponse therapeutique. Dans les autres encephalites virales, la SRM permet de faire le bilan de la gravite et de l'extension des lesions.

International audience; Autophosphorylation at tyrosine is a common process in eukaryotic kinases, which is generally modulated by regulatory ligands and affects the properties of these enzymes. We report that this type of modification occurs also in bacteria, namely in an 81 kDa protein from Acinetobacter johnsonii. This protein is phosphorylated at the expense of ATP exclusively at tyrosine residues. It is located in the inner-membrane fraction of cells and can be totally solubilized by detergents. It has been purified to homogeneity by antiphosphotyrosine immunochromatography. Analysis of the peptides released under trypsin proteolysis of the protein has shown that it autophosphorylates at several tyrosine residues. The discovery of protein autophosphorylation in bacteria seems of special interest for studying the regulatory aspects of this modification when considering the relative simplicity of the bacterial systems, as compared with most eukaryotic systems, namely in terms of physiology and genetics.Autophosphorylation at tyrosine is a common process in eukaryotic kinases, which is generally modulated by regulatory ligands and affects the properties of these enzymes. We report that this type of modification occurs also in bacteria, namely in an 81 kDa protein from Acinetobacter johnsonii. This protein is phosphorylated at the expense of ATP exclusively at tyrosine residues. It is located in the inner-membrane fraction of cells and can be totally solubilized by detergents. It has been purified to homogeneity by antiphosphotyrosine immunochromatography. Analysis of the peptides released under trypsin proteolysis of the protein has shown that it autophosphorylates at several tyrosine residues. The discovery of protein autophosphorylation in bacteria seems of special interest for studying the regulatory aspects of this modification when considering the relative simplicity of the bacterial systems, as compared with most eukaryotic systems, namely in terms of physiology and genetics.

Regulation of the eukaryotic cell cycle involves calcium- and lipid-stimulated kinases acting on cytoskeletal structures; there are two principal reasons for supposing that the regulation of the prokaryotic cell cycle may be fundamentally the same. First, evidence for their fundamental difference is still missing and, second, evidence for prokaryotic homologues of eukaryotic cell cycle proteins is accumulating. Such proteins include those involved in calcium regulation, such as calmodulin and calcium-dependent kinases, and those involved in lipid regulation, such as protein kinase C. Proteins identified as candidates for cytoskeletal elements now include MukB, a putative contractile protein responsible for chromosome segregation, and FtsZ, the key constituent of the "cytokinetic" ring. These similarities allow the application of powerful prokaryotic model systems to one of biology's most profound, complex and urgent problems: the nature of the regulation of the eukaryotic cell cycle.

Reversed-phase liquid chromatography was used as an alternative method for the characterization of the precursor and activated forms of porcine and human pancreatic colipase. Using a Beckman Ultrasphere column with an increasing acetonitrile gradient in 0.1% trifluoroacetic acid, it was possible to obtain well-resolved separation of the precursor form of colipase (procolipase) from its trypsin-activated derivative. This protocol was used (1) to study the activation of porcine procolipase by trypsin or thrombin in vitro, (2) to assess the homogeneity of porcine colipase preparations used in tridimensional structure studies and in combination with immunoaffinity chromatography, (3) to identify the form of colipase present in samples of human pancreatic juice.

Exercise-induced changes in phosphorus-containing metabolites and intracellular pH (pHi) have been studied in the finger flexor muscles of 3 patients with glycogen phosphorylase deficiency (McArdle's disease) in comparison to 14 healthy volunteers. At rest, no difference was observed for PCr/Pi ratio and pHi while patients exhibited a higher PCr/ATP ratio (5.91 +/- 0.98 vs 4.02 +/- 0.6). At end-of-exercise, PCr/Pi was abnormally low (0.51 +/- 0.19 vs 1.64 +/- 0.37) whereas no acidosis was observed. The slow recovery of PCr/Pi ratio indicates an impairment of oxidative capacity accompanying the defect in the glycogenolytic pathway. The failure to observe a transient Pi disappearance at the onset of recovery (an index of glycogen phosphorylase activity) can be used in conjunction with the lack of exercise acidosis as a diagnostic index of McArdle's disease.

Small unilamellar liposomes containing fluorinated steroids (flumethasone and dexamethasone) were obtained. A physicochemical evaluation was conducted based on photon correlation spectroscopy (PCS) and fluorine-19 (19F) nuclear magnetic resonance (NMR) spectroscopy compared with standard biochemical methods (HPLC). The PCS method provides a fast and adequate evaluation of some critical features of liposomes (size, physical stability). In addition, 19F NMR spectroscopy gives substantial information, in a nondestructive manner, on steroid behavior in the membrane upon encapsulation and also when the temperature of liposomes is increased. The combined spectroscopic approach proposed here might prove useful in (1) the management of liposomal formulation, especially in the documentation of physicochemical properties, (2) pharmaceutical control in the industrial production line, and (3) control preceding injection at the clinical site. Spectroscopic techniques might offer a complementary approach to classical biochemical methods in the evaluation of the properties of a liposomal formulation and could be usefully integrated into quality control procedures.

Protein phosphorylation was investigated in the bacterium Acinetobacter calcoaceticus both in vivo and in vitro. In cells grown with [32P]orthophosphate, several radioactive phosphoproteins were detected by gel electrophoresis and autoradiography. These proteins were shown to contain phosphoserine, phosphothreonine, and a relatively large proportion of phosphotyrosine residues. Incubation of cellular extracts with [gamma-32P] ATP also resulted in the phosphorylation of several proteins. At least four of them, namely an 81-kDa protein, were modified at tyrosine. No protein labeling occurred when extracts were incubated with [gamma-32P] ATP or [14C]ATP. Moreover, phosphoproteins were insensitive to snake venom phosphodiesterase. All together these results indicate that A. calcoaceticus harbors different protein kinases including a protein-tyrosine kinase activity. Further analysis of this activity showed that it has little, if any, functional similarity with eukaryotic protein-tyrosine kinases.Protein phosphorylation was investigated in the bacterium Acinetobacter calcoaceticus both in vivo and in vitro. In cells grown with [32P]orthophosphate, several radioactive phosphoproteins were detected by gel electrophoresis and autoradiography. These proteins were shown to contain phosphoserine, phosphothreonine, and a relatively large proportion of phosphotyrosine residues. Incubation of cellular extracts with [gamma-32P] ATP also resulted in the phosphorylation of several proteins. At least four of them, namely an 81-kDa protein, were modified at tyrosine. No protein labeling occurred when extracts were incubated with [gamma-32P] ATP or [14C]ATP. Moreover, phosphoproteins were insensitive to snake venom phosphodiesterase. All together these results indicate that A. calcoaceticus harbors different protein kinases including a protein-tyrosine kinase activity. Further analysis of this activity showed that it has little, if any, functional similarity with eukaryotic protein-tyrosine kinases.

BackgroundDiabetic cardiomyopathy alterations include the presence of interstitial fibrosis. Diabetes also increases the occurrence of arrhythmias, but the pathophysiology remains unclear.PurposeTo describe a non-invasive method using high-resolution myocardial T2 time measurement to assess myocardial fibrosis in diabetic mice in vivo and to correlate this fibrosis with ventricular arrhythmias.MethodsCine-FLASH sequences for morphology and function were followed by two multi-slice spin-echo sequences for T2 time assessment, respectively at 20 and 9 ms echo time (resolution 85×85μm2, slice thickness 1.0 mm, imaging time 15 minutes), in ten 16-week old C57Bl/6J after 8 weeks of streptozotocin-induced diabetes, and ten control mice, under isoflurane anesthesia, on a 11.75T magnet. Programmed stimulation was then realized to assess atrial and ventricular inducibility in both groups. MRI measurements were compared with histological quantification of collagen deposits using picrosirius red staining.ResultsT2 time was lower in diabetic mice (13.8±2.8 ms versus 18.9±2.3 ms in the control group ; p < 0.05). This was associated with a significant increase in collagen deposits, as evaluated by picrosirius red staining, in diabetic mice. Morphologic and functional analysis showed no difference in terms of ejection fraction (60.70±5% versus 60.35±4%) between the two groups, but end-systolic(1.28±0.26 μL/g versus 1.04±0.24 μL/g) and end-diastolic volumes (3.22±0.60 μL/g versus 2.67±0.65μL/g) were significantly increased in the diabetic group. During the electrophysiological study, 3 non sustained ventricular tachycardias were induced in diabetic mice (none in the control group) and 4 supra-ventricular arrhythmias (none in the control group).ConclusionIn diabetic cardiomyopathy, T2 assessment can detect the presence of fibrosis at an early stage. Myocardial fibrosis is a potential substrate for the genesis of (supra)-ventricular arrhythmias in diabetes mellitus.

Histidine-tag (His-tag) is the most frequently used tag to label and purify recombinant protein kinases, namely autokinases. However, when analyzing protein phosphorylation, it appears that this modification occurs not only on the kinase itself but also on several serine residues present in the vector-derived His-tag sequence. These parasite modifications can thus lead to misinterpretation of the data concerning protein phosphorylation. We report here on a modified vector devoid of serine residues in the tag and, therefore, more appropriate and secure for studying protein phosphorylation.Histidine-tag (His-tag) is the most frequently used tag to label and purify recombinant protein kinases, namely autokinases. However, when analyzing protein phosphorylation, it appears that this modification occurs not only on the kinase itself but also on several serine residues present in the vector-derived His-tag sequence. These parasite modifications can thus lead to misinterpretation of the data concerning protein phosphorylation. We report here on a modified vector devoid of serine residues in the tag and, therefore, more appropriate and secure for studying protein phosphorylation.

The complete nucleotide sequence (2386 bp) of the intergenic region between aceK and icIR within the acetate operon of Escherichia coli has been determined. This region contains an open reading frame of 1836 bp whose expression has been detected by protein fusion experiments.

Aspects of energetic and intermediary metabolism were studied in a colon adenocarcinoma cell line (HT29) by multinuclear magnetic resonance spectroscopy. Experiments were carried out on the HT29-D4 clone, which was isolated by limit dilution techniques. This clone, usually undifferentiated (D4-UD), can be maintained in a differentiated state (D4-D) in a glucose-free medium. Metabolic data were obtained by NMR analysis of perchloric acid extracts from D4-UD and D4-D cells. Phosphorus-31 and proton NMR spectra showed the presence of a large amount of choline and phosphorylcholine in the differentiated state (400% and 200%, respectively, of the levels found in D4-UD cells). Other differences appeared in the content of phosphocreatine (absent in D4-D cells) and myoinositol (absent in D4-UD cells). Carbon-13 spectra were recorded from perchloric acid extracts of cells incubated with [1- 13 C]-labeled glucose or [2- 13 C]-labeled acetate. The data indicated that both types of cells metabolize glucose through the glycolytic pathway to give lactate, but only D4-D cells were able to store glucose as glycogen at a very high level. A mathematical analysis of fluxes through the tricarboxylic acid (TCA) cycle was developed on the basis of models derived from previous 14 C tracer studies. The model was based on the steady-state labeling of glutamate carbons by the 13 C isotope and gave the fraction of labeled acetyl-Coa entering the TCA cycle, and the activity of anaplerotic reactions relative to the flux through the citrate synthetase reaction. The data indicated that y > 0.3 in all cases. Only 15% and 30% of labeled acetyl CoA entered the TCA cycle in D4-UD and D4-D cells, respectively, under labeled glucose incubation: these values were significantly different upon labeled acetate feeding, reaching 55% for D4-UD cells and 85% for D4-D cells. The main result of this study is that the process of differentiation of HT29 cells is correlated with a large increase in the activity of oxidative metabolism.

Resume La region des protons aromatiques du spectre de la colipase pancreatique de porc a ete etudiee a 360 MHz entre pH 2 et 12. Les resonances associees aux 26 protons des cycles aromatiques des 2 histidines, 2 phenylalanines et 3 tyrosines ont ete identifiees et des attributions univoques a des residus specifiques sont proposees. L'etude de la variation des deplacements chimiques en fonction du pH a fourni les valeurs de pKa apparents de 7,9 pour His I (His 30), 6,9 pour His II (His 86), 10,4 pour Tyr I (Tyr 56 ou 57), 10,3 pour Tyr II (Tyr 56 ou 57) et 11,3 pour Tyr III (Tyr 53). La valeur elevee du pKa du residu His 30 est attribuable a la proximite du residu Asp 31. La rotation du cycle aromatique du residu Tyr 53 autour de l'axe C β -C γ est genee sur l'echelle des temps de la R.M.N. et sa vitesse de rotation est inferieure a 500 fois/sec a 40°C. Dans les memes conditions, les cycles des 2 autres residus de Tyrosine et des 2 phenylalanines sont en libre rotation. L'etude des perturbations dans le profil des courbes de titrage et la valeur anormale de certains deplacements chimiques ont permis de mettre en evidence une interaction specifique entre His 86 et Tyr I. Le residu Tyr II est egalement affecte mais a un degre moindre. L'existence de cette interaction indique que le repliement de la chaine polypeptidique entraine la proximite spatiale des regions 49–59 et 81–90 de la sequence covalente pour former un site « hydrophobe-aromatiquequi pourrait etre implique dans la reconnaissance des interfaces par la colipase pancreatique.

CD studies have been carried out on horse pancreatic colipases which contain a tryptophan in place of the phenylalanine found at position 52 in the pig protein. From the CD spectra of native proteins in the far ultraviolet region, it was estimated that the secondary structure consists of equal amounts of β-structure and aperiodic arrangements. Colipases contain no α-helical structure. The CD spectra of horse and pig colipases display a positive band at 226 nm the pH dependence of which is characteristic of aromatic chromophores. The near ultraviolet region of the CD spectra of horse and pig colipases contains well-resolved bands at 283–284 nm and 277–278 nm, which corresponds to the tyrosines, with one band at 266–268 nm and one shoulder at 261–263 nm reflecting the contribution of the phenylalanine residues. Comparative analysis of the contribution of sidechains in the aromatic region of the spectra is consistent with a lower solvent accessibility of the lone tryptophan of horse colipase B as compared to isocolipase A. Study of the near ultraviolet CD spectrum of colipase B in the presence of micellar concentration of sodium taurodeoxycholate reveals that the tryptophan (Trp 52 ) moves towards a more hydrophobic environment upon the formation of the colipase-bile salt complex. These results support the conclusion that this residue belongs to the previously identified hydrophobic domain of the colipase molecule which interacts with organized lipids (lipid binding site). The conservative substitution of this residue for a phenylalanine in the pig protein suggests that the aromatic structure might be of critical importance for the binding of colipase to the lipid-water interface.

The secondary structure of porcine colipase (93 residues) was established according to the predictive method of Chou and Fasman (Chou, P.Y. and Fasman, G.D. (1974) Biochemistry 13, 211--222 and 222--245). The relative composition of the conformational regions was as follows: 5% alpha-helix (region 39--44), 25% beta-sheet (three regions, 7--11, 49--57 and 77--85) and eight beta-turns corresponding to 32% of the polypeptide. Colipase contains a large proportion (about 35%) of unordered structure. Estimated values for the alpha-helix and beta-sheet contents from the circular dichroism spectrum were in good accordance with the predicted model. A less satisfactory value was found for the beta-turns. A characteristic feature of the far ultraviolet dichroic spectrum is the presence of an unusual positive band at 225 nm that might be indicative of a particular spatial arrangement of the chromophores in the molecule. Two tyrosines (Tyr56 and Tyr57) and one histidine (His86) are at close vicinity in the three dimensional structure of the protein as shown by proton NMR studies. These residues are located at the end of two beta-sheet hydrophobic regions(49--57 and 77--85) which might play a role in the association of colipase with the lipid-water interface as indicated by results of the NMR studies of the taurodeoxycholate-colipase complex.

In recent years evidence has accumulated that, contrary to an early concept, protein phosphorylation is not restricted to eukaryotic systems but occurs in bacteria as well.

The ability of nifedipine to enhance myocardial protection was assessed on isolated perfused rat hearts subjected to 180 min of hypothermic (20 degrees C), global ischemia, followed by 45 min of normothermic reperfusion. Intracellular pH, ATP, Pi and phosphocreatine content were serially measured at 4 min intervals by phosphorus-31 nuclear magnetic resonance spectroscopy and correlated with simultaneously recorded hemodynamic parameters. Addition of nifedipine (0.075 mumol/l and 0.5 mumol/l) to Saint Thomas' cardioplegic solution reduced Pi accumulation during ischemic arrest and increased phosphocreatine levels during reperfusion. Post-ischemic functional recovery was not improved at a drug concentration of 0.075 mumol/l and was depressed at 0.5 mumol/l. These results clearly show that the presence of nifedipine in Saint Thomas' cardioplegic solution does not provide significant additional myocardial protection under hypothermic conditions.

Summary Polysomes which are maintained in various relaxed mutants of E. coli under aminoacyl-tRNA deprivation are dynamic structures continuously degraded and re-assembled. This was shown by studying, firstly, the decay of polysomes in cells treated with rifampicin and, secondly, the assembly of polysomes after they had been previously converted into monosomes by starving cells for glucose.

Summary Phosphorus-31 nuclear magnetic resonance has been used to study the post mortem catabolism of high-energy phosphate compounds and the associated intracellular pH variation in pure fast- and slow-twitch rabbit muscles and in rabbit muscle with mixed fiber types. Comparative results from pure fiber types are reported for the first time. Large amounts of glycerophosphoryl choline (14.1 μmol/g fresh tissue) are found in the internal conoidal bundle (ICB), a pure oxidative slow twitch muscle, whereas the m. psoas major (PM), a pure glycolytic fast twitch muscle and the m. gastrocnemius caput medialis (GCM), with mixed fiber types, are devoid of the same metabolite. The total content of phosphorylated metabolites is constant among the three muscle types. The time-dependent post mortem changes in phosphorylated metabolites display the expected rapid drop in phosphocreatine and a simultaneous increase in intracellular inorganic phosphate. However, the ATP level remains constant during more than 2 h. Rate constants for metabolite breakdown and apparent ATPase activity have been determined. The comparative kinetics of intracellular acidosis at 25°C yield rates of 3.3 × 10 −3 pH unit/min for PM, 2.7 × 10 −3 pH unit/min for GCM and 3.0 × 10 −3 pH unit/min for ICB. Initial intracellular pH values are 7.07, 7.20 and 7.02, respectively. Upon aging, the heterogeneity of the P i signal reflects the existence of cellular compartments with different internal pH. The results suggest that the more intense low-pH P i signal arises from the sarcoplasmic reticulum while the less intense resonance would reflect the sarcoplasmic higher pH. The temperature effect on post mortem catabolism in the 15–25°C range has been documented. As expected, phosphocreatine and ATP breakdown increase with temperature but at a higher rate for slow-twitch ICB than for fast-twitch PM.

Resume La resonance magnetique nucleaire du phosphore-31 est utilisee de plus en plus frequemment pour les etudes du metabolisme cellulaire dans des conditions qui respectent l'integrite de la cellule. Les avantages specifiques du noyau de phosphore-31 sont discutes dans le cadre general de la RMN in vivo. Des applications selectionnees (exemples pris dans la litterature et travaux de notre laboratoire) sont presentees pour illustrer l'apport de la RMN du P-31 dans les domaines suivants: identification et quantification des metabolites (concentrations absolues et relatives), mesure du pH intracellulaire, compartimentation cellulaire, determination des flux metaboliques dans des conditions normales et pathologiques, etude in vivo des cinetiques enzymatiques a l'etat stationnaire. La RMN du phosphore-31 est comparee aux autres techniques plus classiques d'investigation. L'avenir et le potentiel de l'etude des systemes biologiques intacts par RMN in vivo ainsi que ses applications cliniques sont evalues et discutes. Le couplage de la RMN focalisee aux techniques de reconstruction d'images conduit a proposer le concept d′ « image fonctionnelle— par opposition a l′ « image anatomiquetraditionnelle — pour illustrer l'impact important de cette technologie nouvelle dans l'etude non destructive des mecanismes cellulaires, aussi bien sur les cellules et les tissus intacts que sur les organes excises et perfuses, les animaux vivants et le corps humain.