Identification of the nicotinamide mononucleotide adenylyltransferase of Trypanosoma cruzi

The intracellular parasite Trypanosoma cruzi is the aetiological agent of Chagas disease, a public health concern with an increasing incidence rate. This increase is due, among other reasons, to the parasite’s drug resistance mechanisms, which require nicotinamide adenine dinucleotide (NAD+). Furthermore, this molecule is involved in metabolic and intracellular signalling processes necessary for the survival of T. cruzi throughout its life cycle. NAD+ biosynthesis is performed by de novo and salvage pathways, which converge on the step that is catalysed by the enzyme nicotinamide mononucleotide adenylyltransferase (NMNAT) (enzyme commission number: 2.7.7.1). The identification of the NMNAT of T. cruzi is important for the development of future therapeutic strategies to treat Chagas disease. In this study, a hypothetical open reading frame (ORF) for NMNAT was identified in the genome of T. cruzi. The corresponding putative protein was analysed by simulating structural models. The ORF was amplified from genomic DNA by polymerase chain reaction and was further used for the construction of a corresponding recombinant expression vector. The expressed recombinant protein was partially purified and its activity was evaluated using enzymatic assays. These results comprise the first identification of an NMNAT in T. cruzi using bioinformatics and experimental tools and hence represent the first step to understanding NAD+ metabolism in these parasites.

Erythrocyte glucose-6-phosphate dehydrogenase from Brazilian opossum Didelphis marsupialis

In a comparative study of erythrocyte metabolism of vertebrates, the specific activity of glucose-6-phosphate dehydrogenase (G6PD) of the Brazilian opossum Didelphis marsupialis in a hemolysate was shown to be high, 207 ± 38 IU g-1 Hb-1 min-1 at 37ºC, compared to the human erythrocyte activity of 12 ± 2 IU g-1 Hb-1 min-1 at 37ºC. The apparent high specific activity of the mixture led us to investigate the physicochemical properties of the opossum enzyme. We report that reduced glutathione (GSH) in the erythrocytes was only 50% higher than in human erythrocytes, a value lower than expected from the high G6PD activity since GSH is maintained in a reduced state by G6PD activity. The molecular mass, determined by G-200 Sephadex column chromatography at pH 8.0, was 265 kDa, which is essentially the same as that of human G6PD (260 kDa). The Michaelis-Menten constants (Km: 55 µM) for glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate (Km: 3.3 µM) were similar to those of the human enzyme (Km: 50-70 and Km: 2.9-4.4, respectively). A 450-fold purification of the opossum enzyme was achieved and the specific activity of the purified enzyme, 90 IU/mg protein, was actually lower than the 150 IU/mg protein observed for human G6PD. We conclude that G6PD after purification from the hemolysate of D. marsupialis does not have a high specific activity. Thus, it is quite probable that the red cell hyperactivity reported may be explained by increased synthesis of G6PD molecules per unit of hemoglobin or to reduced inactivation in the RBC hemolysate.

Standardization of metachromatic staining method of myofibrillar ATPase activity of myosin to skeletal striated muscle of mules and donkeys

This study aims at standardizing the pre-incubation and incubation pH and temperature used in the metachromatic staining method of myofibrillar ATPase activity of myosin (mATPase) used for asses and mules. Twenty four donkeys and 10 mules, seven females and three males, were used in the study. From each animal, fragments from the Gluteus medius muscle were collected and percutaneous muscle biopsy was performed using a 6.0-mm Bergström-type needle. In addition to the metachromatic staining method of mATPase, the technique of nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR) was also performed to confirm the histochemical data. The histochemical result of mATPase for acidic pre-incubation (pH=4.50) and alkaline incubation (pH=10.50), at a temperature of 37ºC, yielded the best differentiation of fibers stained with toluidine blue. Muscle fibers were identified according to the following colors: type I (oxidative, light blue), type IIA (oxidative-glycolytic, intermediate blue) and type IIX (glycolytic, dark blue). There are no reports in the literature regarding the characterization and distribution of different types of muscle fibers used by donkeys and mules when performing traction work, cargo transportation, endurance sports (horseback riding) and marching competitions. Therefore, this study is the first report on the standardization of the mATPase technique for donkeys and mules.

Regulation of the expression of NADP-malic enzyme by UV-B, red and far-red light in maize seedlings

The induction of nicotinamide adenine dinucleotide phosphate-malic enzyme (NADP-ME) in etiolated maize (Zea mays) seedlings by UV-B and UV-A radiation, and different levels of photosynthetically active radiation (PAR, 400-700 nm) was investigated by measuring changes in activity, protein quantity and RNA levels as a function of intensity and duration of exposure to the different radiations. Under low levels of PAR, exposure to UV-B radiation but not UV-A radiation for 6 to 24 h caused a marked increase in the enzyme levels similar to that observed under high PAR in the absence of UV-B. UV-B treatment of green leaves following a 12-h dark period also caused an increase in NADP-ME expression. Exposure to UV-B radiation for only 5 min resulted in a rapid increase of the enzyme, followed by a more gradual rise with longer exposure up to 6 h. Low levels of red light for 5 min or 6 h were also effective in inducing NADP-ME activity equivalent to that obtained with UV-B radiation. A 5-min exposure to far-red light following UV-B or red light treatment reversed the induction of NADP-ME, and this effect could be eliminated by further treatment with UV-B or red light. These results indicate that physiological levels of UV-B radiation can have a positive effect on the induction of this photosynthetic enzyme. The reducing power and pyruvate generated by the activity of NADP-ME may be used for respiration, in cellular repair processes and as substrates for fatty acid synthesis required for membrane repair.

Effects of sciatic nerve transection on ultrastructure, NADPH-diaphorase reaction and serotonin-, tyrosine hydroxylase-, c-Fos-, glucose transporter 1- and 3-like immunoreactivities in frog dorsal root ganglion

Frogs have been used as an alternative model to study pain mechanisms. Since we did not find any reports on the effects of sciatic nerve transection (SNT) on the ultrastructure and pattern of metabolic substances in frog dorsal root ganglion (DRG) cells, in the present study, 18 adult male frogs (Rana catesbeiana) were divided into three experimental groups: naive (frogs not subjected to surgical manipulation), sham (frogs in which all surgical procedures to expose the sciatic nerve were used except transection of the nerve), and SNT (frogs in which the sciatic nerve was exposed and transected). After 3 days, the bilateral DRG of the sciatic nerve was collected and used for transmission electron microscopy. Immunohistochemistry was used to detect reactivity for glucose transporter (Glut) types 1 and 3, tyrosine hydroxylase, serotonin and c-Fos, as well as nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase). SNT induced more mitochondria with vacuolation in neurons, satellite glial cells (SGCs) with more cytoplasmic extensions emerging from cell bodies, as well as more ribosomes, rough endoplasmic reticulum, intermediate filaments and mitochondria. c-Fos immunoreactivity was found in neuronal nuclei. More neurons and SGCs surrounded by tyrosine hydroxylase-like immunoreactivity were found. No change occurred in serotonin- and Glut1- and Glut3-like immunoreactivity. NADPH-diaphorase occurred in more neurons and SGCs. No sign of SGC proliferation was observed. Since the changes of frog DRG in response to nerve injury are similar to those of mammals, frogs should be a valid experimental model for the study of the effects of SNT, a condition that still has many unanswered questions.

Gene expression profiling analysis of lung adenocarcinoma

The present study screened potential genes related to lung adenocarcinoma, with the aim of further understanding disease pathogenesis. The GSE2514 dataset including 20 lung adenocarcinoma and 19 adjacent normal tissue samples from 10 patients with lung adenocarcinoma aged 45-73 years was downloaded from Gene Expression Omnibus. Differentially expressed genes (DEGs) between the two groups were screened using the t-test. Potential gene functions were predicted using functional and pathway enrichment analysis, and protein-protein interaction (PPI) networks obtained from the STRING database were constructed with Cytoscape. Module analysis of PPI networks was performed through MCODE in Cytoscape. In total, 535 upregulated and 465 downregulated DEGs were identified. These included ATP5D, UQCRC2, UQCR11 and genes encoding nicotinamide adenine dinucleotide (NADH), which are mainly associated with mitochondrial ATP synthesis coupled electron transport, and which were enriched in the oxidative phosphorylation pathway. Other DEGs were associated with DNA replication (PRIM1, MCM3, and RNASEH2A), cell surface receptor-linked signal transduction and the enzyme-linked receptor protein signaling pathway (MAPK1, STAT3, RAF1, and JAK1), and regulation of the cytoskeleton and phosphatidylinositol signaling system (PIP5K1B, PIP5K1C, and PIP4K2B). Our findings suggest that DEGs encoding subunits of NADH, PRIM1, MCM3, MAPK1, STAT3, RAF1, and JAK1 might be associated with the development of lung adenocarcinoma.

NMDA receptor blockade alters the intracellular distribution of neuronal nitric oxide synthase in the superficial layers of the rat superior colliculus

Nitric oxide (NO) is a molecular messenger involved in several events of synaptic plasticity in the central nervous system. Ca2+ influx through the N-methyl-D-aspartate receptor (NMDAR) triggers the synthesis of NO by activating the enzyme neuronal nitric oxide synthase (nNOS) in postsynaptic densities. Therefore, NMDAR and nNOS are part of the intricate scenario of postsynaptic densities. In the present study, we hypothesized that the intracellular distribution of nNOS in the neurons of superior colliculus (SC) superficial layers is an NMDAR activity-dependent process. We used osmotic minipumps to promote chronic blockade of the receptors with the pharmacological agent MK-801 in the SC of 7 adult rats. The effective blockade of NMDAR was assessed by changes in the protein level of the immediate early gene NGFI-A, which is a well-known NMDAR activity-dependent expressing transcription factor. Upon chronic infusion of MK-801, a decrease of 47% in the number of cells expressing NGFI-A was observed in the SC of treated animals. Additionally, the filled dendritic extent by the histochemical product of nicotinamide adenine di-nucleotide phosphate diaphorase was reduced by 45% when compared to the contralateral SC of the same animals and by 64% when compared to the SC of control animals. We conclude that the proper intracellular localization of nNOS in the retinorecipient layers of SC depends on NMDAR activation. These results are consistent with the view that the participation of NO in the physiological and plastic events of the central nervous system might be closely related to an NMDAR activity-dependent function.

Interactions between intracellular Ca2+ stores: Ca2+ released from the NAADP pool potentiates cADPR-induced Ca2+ release

Cells possess multiple intracellular Ca2+-releasing systems. Sea urchin egg homogenates are a well-established model to study intracellular Ca2+ release. In the present study the mechanism of interaction between three intracellular Ca2+ pools, namely the nicotinic acid adenine dinucleotide phosphate (NAADP), the cyclic ADP-ribose (cADPR) and the inositol 1',4',5'-trisphosphate (IP3)-regulated Ca2+ stores, is explored. The data indicate that the NAADP Ca2+ pool could be used to sensitize the cADPR system. In contrast, the IP3 pool was not affected by the Ca2+ released by NAADP. The mechanism of potentiation of the cADPR-induced Ca2+ release, promoted by Ca2+ released from the NAADP pool, is mediated by the mechanism of Ca2+-induced Ca2+ release. These data raise the possibility that the NAADP Ca2+ store may have a role as a regulator of the cellular sensitivity to cADPR.

High doses of riboflavin and the elimination of dietary red meat promote the recovery of some motor functions in Parkinson's disease patients

Abnormal riboflavin status in the absence of a dietary deficiency was detected in 31 consecutive outpatients with Parkinson's disease (PD), while the classical determinants of homocysteine levels (B6, folic acid, and B12) were usually within normal limits. In contrast, only 3 of 10 consecutive outpatients with dementia without previous stroke had abnormal riboflavin status. The data for 12 patients who did not complete 6 months of therapy or did not comply with the proposed treatment paradigm were excluded from analysis. Nineteen PD patients (8 males and 11 females, mean age ± SD = 66.2 ± 8.6 years; 3, 3, 2, 5, and 6 patients in Hoehn and Yahr stages I to V) received riboflavin orally (30 mg every 8 h) plus their usual symptomatic medications and all red meat was eliminated from their diet. After 1 month the riboflavin status of the patients was normalized from 106.4 ± 34.9 to 179.2 ± 23 ng/ml (N = 9). Motor capacity was measured by a modification of the scoring system of Hoehn and Yahr, which reports motor capacity as percent. All 19 patients who completed 6 months of treatment showed improved motor capacity during the first three months and most reached a plateau while 5/19 continued to improve in the 3- to 6-month interval. Their average motor capacity increased from 44 to 71% after 6 months, increasing significantly every month compared with their own pretreatment status (P < 0.001, Wilcoxon signed rank test). Discontinuation of riboflavin for several days did not impair motor capacity and yellowish urine was the only side effect observed. The data show that the proposed treatment improves the clinical condition of PD patients. Riboflavin-sensitive mechanisms involved in PD may include glutathione depletion, cumulative mitochondrial DNA mutations, disturbed mitochondrial protein complexes, and abnormal iron metabolism. More studies are required to identify the mechanisms involved.

Morphological and physiological alterations induced by lactofen in soybean leaves are reduced with nitric oxide

Lactofen is a diphenylether herbicide recommended to control broad-leaved weeds in soybean (Glycine max) fields and its mechanism of action is the inhibition of protoporphyrinogen-IX oxidase (Protox), which acts in the chlorophyll biosynthesis. This inhibition results in an accumulation of protoporphyrin-IX, which leads to the production of reactive oxygen species (ROS) that cause oxidative stress. Consequently, spots, wrinkling and leaf burn may occur, resulting in a transitory crop growth interruption. However, nitric oxide (NO) acts as an antioxidant in direct ROS scavenging. Thus, the aim of this work was to verify, through phytometric and biochemical evaluations, the protective effect of NO in soybean plants treated with the herbicide lactofen. Soybean plants were pre-treated with different levels of sodium nitroprusside (SNP), a NO-donor substance, and then sprayed with 168 g a.i. ha-1 lactofen. Pre-treatment with SNP was beneficial because NO decreased the injury symptoms caused by lactofen in young leaflets and kept low the soluble sugar levels. Nevertheless, NO caused slower plant growth, which indicates that further studies are needed in order to elucidate the action mechanisms of NO in signaling the stress caused by lactofen in soybean crop.

Angiotensin II stimulates MCP-1 production in rat glomerular endothelial cells via NAD(P)H oxidase-dependent nuclear factor-kappa B signaling

Angiotensin II (Ang II) plays a crucial role in the pathogenesis of renal diseases. The objective of the present study was to investigate the possible inflammatory effect of Ang II on glomerular endothelial cells and the underlying mechanism. We isolated and characterized primary cultures of rat glomerular endothelial cells (GECs) and observed that Ang II induced the synthesis of monocyte chemoattractant protein-1 (MCP-1) in GECs as demonstrated by Western blot. Ang II stimulation, at concentrations ranging from 0.1 to 10 µm, of rat GECs induced a rapid increase in the generation of reactive oxygen species as indicated by laser fluoroscopy. The level of p47phox protein, an NAD(P)H oxidase subunit, was also increased by Ang II treatment. These effects of Ang II on GECs were all reduced by diphenyleneiodonium (1.0 µm), an NAD(P)H oxidase inhibitor. Ang II stimulation also promoted the activation of nuclear factor-kappa B (NF-κB). Telmisartan (1.0 µm), an AT1 receptor blocker, blocked all the effects of Ang II on rat GECs. These data suggest that the inhibition of NAD(P)H oxidase-dependent NF-κB signaling reduces the increase in MCP-1 production by GECs induced by Ang II. This may provide a mechanistic basis for the benefits of selective AT1 blockade in dealing with chronic renal disease.

Effect of polyphenol oxidase (PPO) and air treatments on total phenol and tannin content of cocoa nibs

Cocoa flavour is greatly influenced by polyphenols. These compounds undergo a series of transformations during cocoa processing leading to the characteristic cocoa flavour. The use of exogenous polyphenol oxidase (PPO) proved to be useful to reduce polyphenol content in cocoa nibs. The effect of a PPO associated or not with air over total phenol and tannin content was evaluated. Cocoa nibs were autoclaved and treated with a PPO or water in the absence or presence of an air flow for 0.5, 1, 2 and 3 hours. Total phenol content was reduced in PPO or water treatments, but when associated with air there was an increase in phenol content. Tannin content was reduced only by the treatment with water and air.

Reduced schedule of human anti rabies immunization with Fuenzalida & Palacios vaccine

The present study evaluates the humoral and cellular immune responses in 35 volunteers submited to short antirabies vaccination schedules with the Fuenzalida & Palacios vaccine based on the administration of doses on non consecutive days. The volunteers were divided into two groups. The first group received a total number of five doses given on days 0, 4, 7, 20 and 35. The other group received four doses, the first one being a double dose given on day 0 and than three other single doses on days 7, 20 and 35. The evaluation of humoral immune response was carried out by serum neutralization (SN) and indirect immunofluorescense (IIF) tests, while the cellular immune response was evaluated by lymphoblastic transformation assay (LTA) and skin test (ST). According to our results these reduced schedules elicited early and effective humoral and cellulafimmune responses to rabies antigen suggesting that new reduced schedules should be extensively studied in order to give the proper bases to the proposition of changes in the current long-term schedule.

Reduced schedule of human anti-rabies immunization with Fuenzalida & Palacios vaccine: additional data

It was reevaluated a reduced schedule for anti-rabies post-exposure immunization with newborn mice nervous tissue vaccine (Fuenzalida 8c Palacios) in a group of 30 non exposed volunteers. The vaccine was administered by intramuscular injections on days zero, 2, 4, 16 and 27, in the deltoid area. Antibody levels were determinated by a simplified serum neutralization microtest on days zero, 16 and 37. On days 16 and 37 the antibody levels of the whole group was >0.5 IU/ml and >1.0 IU/ml, respectively. The cell mediated immunity was precociously detected (on day 4) by the delayed type hipersensitivity skin test. Our results show that this reduced schedule elicited an early and effective humoral and cellular immune response. However it is necessary other studies with larger groups of vaccinees in order to obtain definitive conclusion.

Desbalanço redox: NADPH oxidase como um alvo terapêutico no manejo cardiovascular

Vários estudos destacam as espécies reativas de oxigênio e nitrogênio (ERONs) como importantes contribuintes na patogênese de numerosas doenças cardiovasculares, incluindo hipertensão, aterosclerose e falência cardíaca. Tais espécies são moléculas altamente bioativas e com vida curta derivadas, principalmente, da redução do oxigênio molecular. O complexo enzimático da NADPH oxidase é a maior fonte dessas espécies reativas na vasculatura. Sob condições fisiológicas, a formação e eliminação destas substâncias aparecem balanceadas na parede vascular. Durante o desbalanço redox, entretanto, há um aumento na atividade da NADPH oxidase e predomínio de agentes pró-oxidantes, superando a capacidade de defesa orgânica antioxidante. Além disso, tal hiperatividade enzimática reduz a biodisponibilidade do óxido nítrico, crucial para a vasodilatação e a manutenção da função vascular normal. Apesar de a NADPH oxidase relacionar-se diretamente à disfunção endotelial, foi primeiramente descrita por sua expressão em fagócitos, onde sua atividade determina a eficácia dos mecanismos de defesa orgânica contra patógenos. As sutis diferenças existentes entre as unidades estruturais das NADPH oxidases, a depender do tipo celular que as expressa, podem ter implicações terapêuticas, permitindo a inibição seletiva do desequilíbrio redox induzido pela NADPH oxidase, sem comprometer, entretanto, sua participação nas vias fisiológicas de sinalização celular que garantem a proteção contra microorganismos.