Kinetic modeling of tricarboxylic acid cycle and glyoxylate bypass in Mycobacterium tuberculosis, and its application to assessment of drug targets

Background  

Targeting persistent tubercule bacilli has become an important challenge in the development of anti-tuberculous drugs. As the glyoxylate bypass is essential for persistent bacilli, interference with it holds the potential for designing new antibacterial drugs. We have developed kinetic models of the tricarboxylic acid cycle and glyoxylate bypass in Escherichia coli and Mycobacterium tuberculosis, and studied the effects of inhibition of various enzymes in the M. tuberculosis model.     

Pyrroloquinoline-quinone: a reactive oxygen species scavenger in bacteria

Transgenic Escherichia coli expressing pyrroloquinoline-quinone (PQQ) synthase gene from Deinococcus radiodurans showed superior survival during Rose Bengal induced oxidative stress. Such cells showed significantly low levels of protein carbonylation as compared to non-transgenic control. In vitro, PQQ reacted with reactive oxygen species with rate constants comparable to other well known antioxidants, producing non-reactive molecular products. PQQ also protected plasmid DNA and proteins from the oxidative damage caused by gamma-irradiation in solution. The data suggest that radioprotective/oxidative stress protective ability of PQQ in bacteria may be consequent to scavenging of reactive oxygen species per se and induction of other free radical scavenging mechanism.     

Interaction of the disaccharide trehalose with a phospholipid bilayer: a molecular dynamics study

The disaccharide trehalose is well known for its bioprotective properties. Produced in large amounts during stress periods in the life of organisms able to survive potentially damaging conditions, trehalose plays its protective role by stabilizing biostructures such as proteins and lipid membranes. In this study, molecular dynamics simulations are used to investigate the interaction of trehalose with a phospholipid bilayer at atomistic resolution. Simulations of the bilayer in the absence and in the presence of trehalose at two different concentrations (1 or 2 molal) are carried out at 325 K and 475 K. The results show that trehalose is able to minimize the disruptive effect of the elevated temperature and stabilize the bilayer structure. At both temperature, trehalose is found to interact directly with the bilayer through hydrogen bonds. However, the water molecules at the bilayer surface are not completely replaced. At high temperature, the protective effect of trehalose is correlated with a significant increase in the number of trehalose-bilayer hydrogen bonds, predominantly through an increase in the number of trehalose molecules bridging three or more lipid molecules.     

Intracellular calcium activity in isolated bovine adrenal chromaffin cells in the presence and absence of 60 Hz magnetic fields

This study examined whether 60 Hz magnetic field (MF) exposure alters intracellular calcium levels ([Ca(2+)](i)) in isolated bovine adrenal chromaffin cells, a classic model of neural responses. [Ca(2+)](i) was monitored by fluorescence video imaging of cells loaded with the calcium indicator fluo-4 during exposures to magnetic flux densities of 0.01, 0.1, 1.0, 1.4, or 2.0 mT. MFs generated by Helmholtz coils constructed from bifilar wire allowed both 60 Hz field and sham exposures. Following a 5 min monitoring period to establish baseline patterns, cells were subjected for 10 min to a 60 Hz MF, sham field or no field. Reference calcium responses and assessment of cell excitability were obtained by the sequential addition of the nicotinic cholinergic receptor agonist dimethylphenylpiperazinium (DMPP) and a depolarizing concentration of KCl. Throughout an 8 day culture period, cells exhibited spontaneous fluctuations in [Ca(2+)](i). Comparisons of the number of cells exhibiting transients, the number and types of calcium transients, as well as the time during monitoring when transients occurred showed no significant differences between MF exposed cells and either sham exposed or nonexposed cells. With respect to the percentage of cells responding to DMPP, differences between 1 and 2 mT exposed cells and both nonexposed and sham exposed cells reached statistical significance during the first day in culture. No statistically significant differences were observed for responses to KCl. In summary, our data indicate that [Ca(2+)](i) in chromaffin cells is unaffected by the specific 60 Hz MF intensities used in this study. On the other hand, plasma membrane nicotinic receptors may be affected in a manner that is important for ligand-receptor interactions.     

K(+) versus Na(+) ions in a K channel selectivity filter: a simulation study

Molecular dynamics simulations of a bacterial potassium channel (KcsA) embedded in a phospholipid bilayer reveal significant differences in interactions of the selectivity filter with K(+) compared with Na(+) ions. K(+) ions and water molecules within the filter undergo concerted single-file motion in which they translocate between adjacent sites within the filter on a nanosecond timescale. In contrast, Na(+) ions remain bound to sites within the filter and do not exhibit translocation on a nanosecond timescale. Furthermore, entry of a K(+) ion into the filter from the extracellular mouth is observed, whereas this does not occur for a Na(+) ion. Whereas K(+) ions prefer to sit within a cage of eight oxygen atoms of the filter, Na(+) ions prefer to interact with a ring of four oxygen atoms plus two water molecules. These differences in interactions in the selectivity filter may contribute to the selectivity of KcsA for K(+) ions (in addition to the differences in dehydration energy between K(+) and Na(+)) and the block of KcsA by internal Na(+) ions. In our simulations the selectivity filter exhibits significant flexibility in response to changes in ion/protein interactions, with a somewhat greater distortion induced by Na(+) than by K(+) ions.     

SIMILARITIES OF β-BUNGAROTOXIN AND PHOSPHOLIPASE A2 AND THEIR MECHANISM OF ACTION

Abstract— β-Bungarotoxin, a presynaptic neurotoxin isolated from the venom of Bungarus multicinctus , has been shown to initially cause an increase in the frequency of miniature endplate potentials and subsequently block neuromuscular transmission by inhibiting nerve impulse induced release of acetylcholine. In rat brain synaptosomes it causes a Ca²⁺-dependent release of acetylcholine together, with a strong inhibition of the high affinity choline uptake system. In this report we demonstrate that β-bungarotoxin acts as a phospholipase A₂ (phosphatide 2-acyl hydrolase, EC 3.1.1.4), liberating fatty acids from synaptic membrane phospholipids. It also exhibits a striking similarity in a number of neurochemical properties with that of a purified phospholipase A₂ from Naja naja siamensis. In addition, both agents produce a marked depolarization of synaptosomal preparations as measured by a fluorescent dye. We propose that disruption of the membrane phospholipids by phospholipase activity can lead to depolarization of the synaptosomal preparation which promotes both transmitter release and inhibition of the energy-dependent high affinity choline uptake system. With this decreased supply of choline, the acetylcholine content of the cell would be gradually depleted leading to a decrease in transmission.     

Metabolic effects of hydrocortisone in mouse brain

The effect of intramuscular administration of hydrocortisone (10 mg/day per animal) for 5 days has been studied on the content of the amino acids belonging to the glutamate family, in the different regions of the mouse brain, along with the activities of glutamine synthetase, glutamate dehydrogenase, and aspartate, alanine, tyrosine, and ornithine aminotransferases. Further, since proline too is related to glutamate metabolism, the activity of proline oxidase was also studied in these regions. As hydrocortisone is known to influence the ionic fluxes in different tissues and the nitrogen metabolism, the activities of Na⁺,K⁺-ATPase together with the content of RNA and protein have also been estimated. A fall in the amino acids of the glutamate family in all three regions was observed with an increase in glutamate dehydrogenase activity in cerebral cortex. A significant fall in the protein content was also observed, mainly in the brain stem. A universal increase in Na⁺,K⁺-ATPase activity was observed in all three regions, with the highest in the cerebral cortex. The results indicate that hydrocortisone triggers increased utilization of glutamate in brain as an alternative to glucose, thereby shifting the nitrogen metabolism toward catabolism. The increased activity of Na⁺,K⁺-ATPase under these conditions would further aggravate the same and may lead to membrane stabilization.     

Acute metabolic effects of ammonia in mouse brain

Acute effects of intraperitoneal administration of ammonium chloride (200 mg/kg) on Na⁺,K⁺-ATPase and amino acid content of the glutamate family (glutamate, aspartate, alanine, glutamine, and GABA), as well as the enzymes involved in the metabolism of these amino acids, have been studied in the different regions of brain and liver in mice. A significant increase in the activity of Na⁺,K⁺-ATPase was observed in the cerebellum, cerebral cortex, and brain stem. A similar increase in the activity of glutamate dehydrogenase was observed in the brain stem, while a moderate increase in the activity of this enzyme was observed in the cerebral cortex and liver in the mice treated with ammonium chloride. In all three regions of brain, a 50% decrease was observed in the activity of alanine aminotransferase, while the activity of aspartate aminotransferase significantly rose in the brain stem. The activity of glutamine synthetase did not change much in the three regions of brain, and a significant fall was registered in the liver. The activity of tyrosine aminotransferase showed a rise in the cerebellum, brain stem, and in liver. Not much change was observed in the protein content in either brain or liver, whereas there was a 1.5-fold increase in the total RNA content in the liver of the animals treated with ammonium chloride. Under the experimental conditions, there was an increase only in the content of glutamine, of all the amino acids tested, in the cerebral cortex and liver. Similar results were obtained with homogenates of tissues enriched with ammonium chloride (in vitro) for the enzyme systems studied. These results are discussed, and the probable metabolic and functional significance of ammonia in brain is indicated.     

Predictive value of the analogy between hormone-sensitive adenylate cyclase and light-sensitive photoreceptor cyclic GMP Phosphodiesterase: A specific role for a light-sensitive GTPase as a component in the activation sequence

We report experiments which involve a light sensitive GTPase in the light dependent activation of retinal rod 3′5′-cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE). The data suggest that the light activated GTPase is intermediate between rhodopsin and PDE in the light-dependent activation sequence. We list the many striking similarities between hormone sensitive adenylate cyclase and light activated PDE in order to emphasize that the findings presented herein may have predictive value for ongoing studies of the hormone sensitive adenylate cyclase specifically regarding the role of the hormone activated GTPase in the activation sequence.