Defective ATP breakdown activity related to an ENTPD1 gene mutation demonstrated using 31P NMR spectroscopy

Quantification of the ENTPDase activity of human mononuclear cells by 31P NMR spectroscopy and the effect of a stop-codon mutation in the ENTPD1 gene.

Preliminary study using visible and SW-NIR analysis for evaluating the loss of freshness in commercially packaged cooked ham and Turkey ham

A non-destructive Vis-NIR spectroscopy (400–1000 nm) method was developed to evaluate the loss of freshness of sliced and commercially packaged cooked ham and turkey ham without any sample manipulation. The spectra were recorded at 0, 30, 40, and 60 days using a camera, spectral filter (400–1000 nm) and a halogen floodlighting system which had been were developed and calibrated for the purpose. Physico-chemical, biochemical, and microbiological properties such as pH, total volatile basic nitrogen (TVB-N), ATP breakdown compounds, and colony-forming units were determined to predict the degradation of freshness. The image spectra obtained from visible and SW-NIR  spectroscopy were related to the storage time of the samples. A PLS-DA model was developed independently for packaged or unpackaged samples using the second derivative of the spectra. Mean R² prediction obtained for cooked ham was 0.915 and 0.949 for Turkey ham. The technique developed could be applied to monitoring the freshness of commercial packed cooked ham and turkey ham as a non-destructive technique. Further studies will be needed to check the spectra obtained from samples of different commercial brands in order to evaluate more precisely the efficiency of the method.

Isolation and characterization of pigeon breast muscle cytosolic 5'-nucleotidase-I (cN-I).

5'-Nucleotidase specific towards dCMP and AMP was isolated from avian breast muscle and characterized. It was found to be similar to a type-I form (cN-I) identified earlier as the AMP-selective 5'-nucleotidase responsible for adenosine formation during ATP breakdown in transfected COS-7 cells. Expression pattern of the cN-I gene in pigeon tissues indicated breast muscle as a rich source of the transcript. We purified the enzyme from this source using two-step chromatography and obtained an active homogenous preparation, free of ecto-5'-nucleotidase activity. The tissue content of the activity was calculated at 0.09 U/g wet weight. The specific activity of the enzyme preparation was 4.33 U/mg protein and it preferred dCMP and AMP to dAMP and IMP as a substrate. Its kinetic properties were very similar to those of the enzyme purified earlier from heart tissue. It was strongly activated by ADP. Inhibition by inorganic phosphate was more pronounced than in heart-isolated cN-I. Despite this difference, a similar physiological function is suggested for cN-I in both types of muscle.

Mechanism of active repolarization of inhibitory junction potential in murine colon

Enteric inhibitory responses in gastrointestinal (GI) smooth muscles involve membrane hyperpolarization that transiently reduce the excitability of GI muscles. We examined the possibility that an active repolarization mechanism participates in the restoration of resting membrane potential after fast inhibitory junction potentials (IJPs) in the murine colon. Previously, we showed these cells express a voltage-dependent nonselective cation conductance (NSCC) that might participate in active repolarization of IJPs. Colonic smooth muscle cells were impaled with micro-electrodes and voltage responses to nerve-evoked IJPs, and locally applied ATP were recorded. Ba2+ (500 μM), a blocker of the NSCC, slowed the rate of repolarization of IJPs. We also tested the effects of Ba2+, Ni2+, and mibefradil, all blockers of the NSCC, on responses to locally applied ATP. Spritzes of ATP caused transient hyperpolarization, and the durations of these responses were significantly increased by the blockers of the NSCC. We considered whether NSCC blockers might affect ATP metabolism and found that Ni2+ decreased ATP breakdown in colonic muscles. Mibefradil had no effect on ATP metabolism. Because both Ni2+ and mibefradil had similar effects on prolonging responses to ATP, it appears that restoration of resting membrane potential after ATP spritzes is not primarily due to ATP metabolism. Neurally released enteric inhibitory transmitter and locally applied ATP resulted in transient hyperpolarizations of murine colonic muscles. Recovery of membrane potential after these responses appears to involve an active repolarization mechanism due to activation of the voltage-dependent NSCC expressed by these cells.

Effects of Hypothermia on Energy Metabolism in Mammalian Central Nervous System

This review analyzes, in some depth, results of studies on the effect of lowered temperatures on cerebral energy metabolism in animals under normal conditions and in some selected pathologic situations. In sedated and paralyzed mammals, acute uncomplicated 0.5- to 3-h hypothermia decreases the global cerebral metabolic rate for glucose (CMRglc) and oxygen (CMRO2) but maintains a slightly better energy level, which indicates that ATP breakdown is reduced more than its synthesis. Intracellular alkalinization stimulates glycolysis and independently enhances energy generation. Lowering of temperature during hypoxia–ischemia slows the rate of glucose, phosphocreatine, and ATP breakdown and lactate and inorganic phosphate formation, and improves recovery of energetic parameters during reperfusion. Mild hypothermia of 12 to 24-h duration after normothermic hypoxic–ischemic insults seems to prevent or ameliorate secondary failures in energy parameters. The authors conclude that lowered head temperatures help to protect and maintain normal CNS function by preserving brain ATP supply and level. Hypothermia may thus prove a promising avenue in the treatment of stroke and trauma and, in particular, of perinatal brain injury.

Mechanisms of reduced SR Ca2+ release induced by inorganic phosphate in rat skeletal muscle fibers

The effects of inorganic phosphate (Pi) on Ca2+ release from the sarcoplasmic reticulum (SR) were studied in mechanically skinned rat skeletal muscle fibers. Application of caffeine or T-tubule depolarization was used to induce Ca2+ release from the SR, which was detected using fura 2 fluorescence. Addition of Pi (1–40 mM) caused a reversible and concentration-dependent reduction in the caffeine-induced Ca2+ transient. This effect was apparent at low Pi concentration (<5 mM), which did not result in detectable precipitation of calcium phosphate within the SR. The inhibitory effect of Pi exhibited a marked dependence on free Mg2+ concentration. At 0.5 mM free Mg2+, 5 mM Pi reduced the caffeine-induced transient by 25.1 ± 4.1% ( n = 13). However, at 1.5 mM free Mg2+, 5 mM Pi reduced the amplitude of caffeine-induced Ca2+ transients by 68.9 ± 3.1% ( n = 10). Depolarization-induced SR Ca2+release was similarly affected. These effects of Pi may be important in skeletal muscle fatigue, if an inhibitory action of Pi on SR Ca2+ release is augmented by the rise in cytosolic Mg2+ concentration, which accompanies ATP breakdown.