Construction of Mycobacterium tuberculosis cdd knockout and evaluation of invasion and growth in macrophages.

Cytidine deaminase (MtCDA), encoded by cdd gene (Rv3315c), is the only enzyme identified in nucleotide biosynthesis pathway of Mycobacterium tuberculosis that is able to recycle cytidine and deoxycytidine. An M. tuberculosis knockout strain for cdd gene was obtained by allelic replacement. Evaluation of mRNA expression validated cdd deletion and showed the absence of polar effect. MudPIT LC-MS/MS data indicated thymidine phosphorylase expression was decreased in knockout and complemented strains. The cdd disruption does not affect M. tuberculosis growth both in Mid- dlebrook 7H9 and in RAW 264.7 cells, which indicates that cdd is not important for macrophage invasion and virulence.

Gene replacement and quantitative mass spectrometry approaches validate guanosine monophosphate synthetase as essential for Mycobacterium tuberculosis growth.

Guanosine monophosphate synthetase (GMPS), encoded by guaA gene, is a key enzyme for guanine nucleotide biosynthesis in Mycobacterium tuberculosis. The guaA gene from several bacterial pathogens has been shown to be involved in virulence; however, no information about the physiological effect of direct guaA deletion in M. tuberculosis has been described so far. Here, we demonstrated that the guaA gene is essential for M. tuberculosis H37Rv growth. The lethal phenotype of guaA gene disruption was avoided by insertion of a copy of the ortholog gene from Mycobacterium smegmatis, indicating that this GMPS protein is functional in M. tuberculosis. Protein validation of the guaA essentiality observed by PCR was approached by shotgun proteomic analysis. A quantitative method was performed to evaluate protein expression levels, and to check the origin of common and unique peptides from M. tuberculosis and M. smegmatis GMPS proteins. These results validate GMPS as a molecular target for drug design against M. tuberculosis, and GMPS inhibitors might prove to be useful for future development of new drugs to treat human tuberculosis.

Experimental evidence that pristanic acid disrupts mitochondrial homeostasis in brain of young rats.

Patients affected by peroxisomal disorders commonly present neurologic dysfunction and brain abnormalities, whose neuropathology is poorly understood. Given that high sustained concentrations of pristanic acid (Prist) are found in the brain of these patients, it is conceivable that this complex branched-chain fatty acid is neurotoxic. Therefore, the present work investigated the in vitro effects of Prist at similar concentrations found in plasma of affected patients with some peroxisomal disorders on important parameters of energy homeostasis, including respiratory parameters determined by oxygen consumption, membrane potential (ΔΨm), NAD(P)H content, and swelling in mitochondrial preparations obtained from brain of young rats using glutamate plus malate or succinate as respiratory substrates. Prist markedly increased state 4 respiration and decreased state 3 respiration, the respiratory control ratio (RCR), and the ADP/O ratio with both substrates. The mitochondrial ΔΨm and the matrix NAD(P)H content were also decreased by Prist, which was also able to provoke mitochondrial swelling. Furthermore, Prist-induced mitochondrial swelling was dependent on oxidative damage to the permeability transition pore (PTP), because cyclosporine A and the thiol-reducing agent N-acetylcysteine totally prevented mitochondrial swelling. These data suggest that Prist impairs mitochondrial homeostasis, acting as an uncoupler of oxidative phosphorylation and as a metabolic inhibitor, besides causing mitochondrial swelling probably mediated by the permeability transition pore. It is proposed that these pathomechanisms may potentially be involved in the neurological abnormalities characteristic of the peroxisomal diseases in which Prist accumulates.

Glycine intrastriatal administration induces lipid and protein oxidative damage and alters the enzymatic antioxidant defenses in rat brain.

AIMS: We investigated the effects of in vivo intrastriatal administration of glycine (Gly), which is found at high concentrations in the brain of patients affected by nonketotic hyperglycinemia (NKH), on important parameters of oxidative stress. MAIN METHODS: Thiobarbituric acid-reactive substances values (TBA-RS, lipid peroxidation), carbonyl formation (protein oxidative damage), sulfhydryl content, reduced glutathione concentrations, nitric oxide production and the activities of the antioxidant enzymes glutathione peroxidase, glutathione reductase, catalase, superoxide dismutase and glucose-6-phosphate dehydrogenase (antioxidant defenses) were measured in striatum from 30-day-old rats after Gly injection. KEY FINDINGS: Gly administration significantly increased TBA-RS values, implying lipid oxidative damage. Furthermore, Gly-induced increase of TBA-RS was fully prevented by the NMDA receptor antagonist MK-801, indicating the involvement of the NMDA glutamate receptor in this effect. Gly injection also induced protein carbonyl formation, as well as elevation of the activities of glutathione peroxidase, glutathione reductase, catalase and superoxide dismutase. In contrast, glutathione levels, sulfhydryl content, nitric oxide production and the activity of glucose-6-phosphate dehydrogenase were not modified by Gly. SIGNIFICANCE: The data shows that Gly in vivo administration causes lipid peroxidation, probably secondary to NMDA stimulation, induces protein oxidation and modulates the activities of important antioxidant enzymes in the striatum. In case these findings can be extrapolated to the human NKH, it is feasible that oxidative stress may be involved in the pathophysiology of the brain injury observed in patients with this neurometabolic disease.

Neurochemical evidence that pristanic acid impairs energy production and inhibits synaptic Na(+), K(+)-ATPase activity in brain of young rats.

Pristanic acid (Prist) accumulates in some peroxisomal disorders characterized by neurologic dysfunction and brain abnormalities. The present work investigated the in vitro effects of Prist on important parameters of energy metabolism in brain cortex of young rats. CO(2) production from labeled acetate and the activities of the respiratory chain complexes I-IV, creatine kinase and synaptic Na(+), K(+)-ATPase were measured. Prist decreased CO(2) production and the activities of complexes I, II and II-III. Prist also reduced Na(+), K(+)-ATPase activity, but did not affect the activity of creatine kinase. Considering the importance of the citric acid cycle and the electron flow through the respiratory chain for brain energy production and of Na(+), K(+)-ATPase for the maintenance of membrane potential, the present data indicate that Prist compromises brain bioenergetics and neurotransmission. It is presumed that these pathomechanisms may be involved in the neurological damage found in patients affected by disorders in which Prist accumulates.

Experimental evidence that methylmalonic acid provokes oxidative damage and compromises antioxidant defenses in nerve terminal and striatum of young rats.

Methylmalonic acidemia and propionic acidemia are organic acidemias biochemically characterized by predominant tissue accumulation of methylmalonic acid (MMA) and propionic acid (PA), respectively. Affected patients present predominantly neurological symptoms, whose pathogenesis is not yet fully established. In the present study we investigated the in vitro effects of MMA and PA on important parameters of lipid and protein oxidative damage and on the production of reactive species in synaptosomes from cerebrum of developing rats. Synaptosomes correspond to nerve terminals that have been used to investigate toxic properties of compounds on neuronal cells. The in vivo effects of intrastriatal injection of MMA and PA on the same parameters and on enzymatic antioxidant defenses, were also studied. MMA-induced in vitro and in vivo lipid peroxidation and protein oxidative damage. Furthermore, the lipid oxidative damage was attenuated or prevented, pending on the doses utilized, by the free radical scavengers α-tocopherol, melatonin and by the NMDA glutamate receptor antagonist MK-801, implying the involvement of reactive species and glutamate receptor activation in these effects. In addition, 2',7'-dichlorofluorescein diacetate oxidation was significantly increased in synaptosomes by MMA, reinforcing that reactive species generation is elicited by this organic acid. We also verified that glutathione peroxidase activity was inhibited by intrastriatal MMA injection. In contrast, PA did not induce any significant effect on all parameters examined in vitro and in vivo, implying a selective action for MMA. The present data demonstrate that oxidative stress is induced by MMA in vitro in nerve terminals and in vivo in striatum, suggesting the participation of neuronal cells in MMA-elicited oxidative damage.

Soybean and sunflower oil-induced insulin resistance correlates with impaired GLUT4 protein expression and translocation specifically in white adipose tissue.

Free fatty acids are known for playing a crucial role in the development of insulin resistance. High fat intake is known for impairing insulin sensitivity; however, the effect of vegetable-oil injections have never been investigated. The present study investigated the effects of daily subcutaneous injections (100 microL) of soybean (SB) and sunflower (SF) oils, during 7 days. Both treated groups developed insulin resistance as assessed by insulin tolerance test. The mechanism underlying the SB- and SF-induced insulin resistance was shown to involve GLUT4. In SB- and SF-treated animals, the GLUT4 protein expression was reduced approximately 20% and 10 min after an acute in vivo stimulus with insulin, the plasma membrane GLUT4 content was approximately 60% lower in white adipose tissue (WAT). No effects were observed in skeletal muscle. Additionally, both oil treatments increased mainly the content of palmitic acid ( approximately 150%) in WAT, which can contribute to explain the GLUT4 regulations. Altogether, the present study collects evidence that those oil treatments might generate insulin resistance by targeting GLUT4 expression and translocation specifically in WAT. These alterations are likely to be caused due to the specific local increase in saturated fatty acids that occurred as a consequence of oil daily injections.

Seasonal variation in glucose and neutral amino acid uptake in the estuarine crab Neohelice granulata.

This study investigates the mechanisms of glucose and amino acid transport in gills and jaw muscle of N. granulata collected from an estuarine natural population. The physicochemical parameters of the estuarine environment and of this crustacean's hemolymph were measured during different seasons of the year. In summer, the lagoon water osmolality increased (5-6 times), and hemolymph osmolality decreased. In fall, water pH increased, whereas hemolymph pH decreased markedly. In all seasons, 2-deoxi glucose (DG) uptake in gills was significantly higher than 3-O methyl-glucose (MG) uptake. Phloretin reduced DG uptake in gills; phloretin and phlorizin did not affect MG uptake in this organ. DG and MG uptake was highest in gills during spring and summer. In jaw muscle, MG uptake in winter and spring was higher than DG uptake. In fall, gill methyl aminoisobutyric acid (MeAIB) uptake increased. In jaw muscle, MeAIB uptake was higher in spring. The observed changes in glucose uptake and in the type of glucose and amino acid transporter used in gills and muscle appear to be strategies used by N. granulata to minimize seasonal oscillations in the environmental parameters of their estuarine habitat.

Hepatopancreas gluconeogenesis during anoxia and post-anoxia recovery in Chasmagnathus granulata crabs maintained on high-protein or carbohydrate-rich diets.

C. granulata is a semiterrestrial crab that lives in the mesolittoral and the supralittoral zones of estuaries and faces hypoxia and anoxia when exposed to atmospheric air. The carbohydrate or protein content of the diets administered to the crabs induced different metabolic adjustments during anoxia and post-anoxia recovery period. During the first hour in anoxia a marked increase in L-lactate concentration in hemolymph was induced, followed by a reduction in its levels accompanied by two peaks in hepatopancreas gluconeogenic capacity. Anoxia exposure did not induce a reduction in the hepatopancreas phosphoenolpyruvate carboxykinase activity in either dietary group. Our results suggest that in anaerobiosis this crab uses the conversion of lactate to glucose in hepatopancreas to maintain the acid-base balance and the glucose supply. In post-anoxia recovery, the fate of L-lactate is the hepatopancreas gluconeogenesis in high protein maintained crabs. On the other hand, in the crabs maintained on carbohydrate-rich diet the L-lactate levels decreased gradually in the hemolymph during the post-anoxia recovery; however, the hepatopancreas gluconeogenesis did not increase. In both dietary groups, an increase in the gluconeogenic capacity of hepatopancreas occurred at 30 h of post-anoxia recovery.