Differential effects of polyamine derivative compounds against Leishmania infantum promastigotes and axenic amastigotes

The natural polyamines are ubiquitous polycationic compounds that play important biological functions in cell growth and differentiation. In the case of protozoan species that are causative agents of important human diseases such as Leishmaniasis, an exogenous supply of polyamines supports parasite proliferation. In the present study, we have investigated the effect of three polyamine derivatives, (namely bis-naphthalimidopropyl putrescine (BNIPPut), spermidine (BNIPSpd) and spermine (BNIPSpm)), on the proliferative stages of Leishmania infantum, the causative agent of visceral leishmaniasis in the Mediterranean basin. A significant reduction of promastigotes and axenic amastigotes growth was observed in the presence of increasing concentrations of the drugs, although the mechanisms leading to the parasite growth arrest seems to be different. Indeed, by using a number of biochemical approaches to analyse the alterations that occurred during early stages of parasite-drug interaction (i.e. membrane phosphatidylserine exposure measured by annexin V binding, DNA fragmentation, deoxynucleotidyltranferase-mediated dUTP end labelin (TUNEL), mitochondrial transmembrane potential loss), we showed that the drugs had the capacity to induce the death of promastigotes by a mechanism that shares many features with metazoan apoptosis. Surprisingly, the amastigotes did not behave in a similar way to promastigotes. The drug inhibitory effect on amastigotes growth and the absence of propidium iodide labelling may suggest that the compounds are acting as cytostatic substances. Although, the mechanisms of action of these compounds have yet to be elucidated, the above data show for the first time that polyamine derivatives may act differentially on the Leishmania parasite stages. Further chemical modifications are needed to make the polyamine derivatives as well as other analogues able to target the amastigote stage of the parasite.     

Chromosome Banding and 18S rDNA in situ Hybridization Analysis of Seven Species of the Family Achiridae (Teleostei: Pleuronectiformes)

Achiridae is an important family of the order Pleuronectiformes widely distributed in North, Central, and South America with freshwater and marine species. In the present study cytogenetic analyses comprising conventional and molecular techniques were carried out in seven species of this family. The following diploid numbers (2n) and fundamental numbers (FN) were obtained: Achirus declivis 2n = 34, FN = 52; Achirus lineatus 2n = 40, FN = 66; Catathyridium jenynsi 2n = 40 and FN = 50; Gymnachirus nudus 2n = 36 and FN = 50; Hypoclinemus mentalis 2n = 38 and FN = 54; Trinectes paulistanus 2n = 42 and FN = 52; and Trinectes sp. 2n = 38 and FN = 54. All species presented a single nucleolar organizer region (NOR) bearing chromosome pair and C-band positive segments mainly distributed at the pericentromeric position. The wide variation observed in chromosome number and FN suggests the occurrence of larger chromosome rearrangements in the family Achiridae if compared with other families of the same order.     

Inhibition of Mitochondrial Respiratory Chain in the Brain of Adult Rats After Acute and Chronic Administration of Methylphenidate

Methylphenidate (MPH) is frequently prescribed for the treatment of attention deficit/hyperactivity disorder. It was previously demonstrated that MPH altered brain metabolic activity. Most cell energy is obtained through oxidative phosphorylation, in the mitochondrial respiratory chain. However, there are still few studies about MPH effects on the brain of adult rats. Thus, in the present study we evaluated the effect of acute or chronic administration of MPH on the activities of mitochondrial respiratory chain complexes I–IV in the brain of adult rats. For acute administration, a single injection of MPH was given to 60-day-old rats. For chronic administration, MPH injections were given to 60-day-old rats once daily for 28 days. Our results showed that complexes I, II, III and IV were inhibited after acute or chronic MPH administration in the hippocampus, prefrontal cortex, striatum and cerebral cortex. On the other hand, cerebellum was not affected.     

Effect of Acute and Chronic Administration of Methylphenidate on Mitochondrial Respiratory Chain in the Brain of Young Rats

Methylphenidate is commonly used for the treatment of attention deficit/hyperactivity disorder. There are still few works regarding the effects of methylphenidate on brain energy metabolism. Thus, in the present study we evaluated the effect of chronic administration of methylphenidate on the activities of mitochondrial respiratory chain complexes I and III in the brain of young rats. The effect of acute administration of methylphenidate on mitochondrial respiratory chain complexes I, II, III and IV in the brain of young rats was also investigated. For acute administration, a single injection of methylphenidate was given to rats on postnatal day 25. For chronic administration, methylphenidate injections were given starting at postnatal day 25 once daily for 28 days. Our results showed that complexes I and III were not affected by chronic administration of methylphenidate. Moreover, the acute administration of methylphenidate decreased complex I activity in cerebellum and prefrontal cortex, whereas complexes II, III and IV were not altered.     

Role of Tonoplast Proton Pumps and Na+/H+ Antiport System in Salt Tolerance of Populus euphratica Oliv.

Populus euphratica has been used as a plant model to study resistance against salt and osmotic stresses, with recent studies having characterized the tonoplast and the plasma membrane ATPases, and two Na⁺/H⁺ antiporters, homologs of the Arabidopsis tonoplast AtNHX1, were published in databases. In the present work we show that P. euphratica suspension-cultured cells are highly tolerant to high salinity, being able to grow with up to 150 mM NaCl in the culture medium without substantial modification of the final population size when compared to the control cells in the absence of salt. At a salt concentration of 300 mM, cells were unable to grow but remained highly viable up to 17 days after subculture. The addition of a 1-M-NaCl pulse to unadapted cells did not promote a significant loss in cell viability within 48 h. In tonoplast vesicles purified from cells cultivated in the absence of salt and from salt-stressed cells, vacuolar H⁺-pyrophosphatase (V-H⁺-PPase) seemed to be the primary tonoplast proton pump; however, there appears to be a decrease in V-H⁺-PPase activity with exposure to NaCl, in contrast to the sodium-induced increase in the activity of vacuolar H⁺-ATPase (V-H⁺-ATPase). Despite reports that in P. euphratica there is no significant difference in the concentration of Na⁺ in the different cell compartments under NaCl stress, in the present study, confocal and epifluorescence microscopic observations using a Na⁺-sensitive probe showed that suspension-cultured cells subject to a salt pulse accumulated Na⁺ in the vacuole when compared with control cells. Concordantly, a tonoplast Na⁺/H⁺ exchange system is described whose activity is upregulated by salt and, indirectly, by a salt-mediated increase of V-H⁺-ATPase activity.     

Effect of Electroconvulsive Shock on Mitochondrial Respiratory Chain in Rat Brain

It is well described that impairment of energy production has been implicated in the pathogenesis of a number of diseases. Although several advances have occurred over the past 20 years concerning the use and administration of electroconvulsive therapy (ECT) to minimize its side effects, little progress has been made in understanding its mechanism of action. In this work, our aim was to measure the activities of mitochondrial respiratory chain complexes II and IV and succinate dehydrogenase from rat brain after acute and chronic electroconvulsive shock (ECS). Our results showed that mitochondrial respiratory chain enzymes activities were increased after acute ECS in hippocampus, striatum and cortex of rats. Besides, we also demonstrated that complex II activity was increased after chronic ECS in cortex, while hippocampus and striatum were not affected. Succinate dehydrogenase, however, was inhibited after chronic ECS in striatum, activated in cortex and not affected in hippocampus. Finally, complex IV was not affected by chronic ECS in hippocampus, striatum and cortex. Our findings demonstrated that brain metabolism is altered by ECS.     

Leptin effect on RANKL and OPG expression in MC3T3-E1 osteoblasts

Recent studies have suggested that leptin hormone may play a pivotal role on bone remodeling through a direct effect by modulating positively the OPG/RANKL balance. Here, we investigate the effect of leptin hormone on RANKL and OPG expression in MC3T3-E1 osteoblasts using RT-PCR and ELISA measurements. We have at first identified the expression of Ob-Rb and Ob-Ra leptin receptor isoforms in MC3T3-E1 and observed that these cells respond to mrleptin treatments. We then investigated the effect of mrleptin on RANKL and OPG expression. We show that mrleptin dose-dependently regulated the expression of RANKL mRNA with complete inhibition observed at concentrations higher than 12 ng/ml. This effect was confirmed with sRANKL protein measurements. However, the exposure of MC3T3-E1 to mrleptin had no effect on OPG mRNA. Taken together, these results suggest that leptin modulates positively OPG/RANKL balance by inhibiting the expression of RANKL gene.     

K+ channels involved in contractility of rabbit small intestine

Most excitable cells, including gastrointestinal smooth muscle cells, express several types of K+ channels. The aim of this study was to examine the types of K' channels involved in the contractility of longitudinal smooth muscle of rabbit small intestine in vitro. Spontaneous contractions and KCl-stimulated contractions were reduced by atropine, phentolamine, propranolol, suramin, tetrodotoxin and indomethacin. The amplitude and tone of spontaneous contractions were increased by apamin, charybdotoxin, iberiotoxin, E4031, tetraetylammonium (TEA) and BaCl2. The frequency of contractions was reduced in the presence of apamin and TEA and increased by charybdotoxin. It was found that 4-aminopyridine increased the tone of spontaneous contractions and reduced the amplitude and frequency of contractions. Glibenclamide did not modify the amplitude, frequency or tone of contractions. KCl-stimulated contractions were increased by E4031, were not modified by apamin, glibenclamide, NS1619 or diazoxide, and were reduced by charybdotoxin, TEA, 4-aminopyridine or BaCl2. These results suggest that both Ca2+-activated K+ channels of small and high conductance, and HERG K+ channels and inward rectifier K+ channels participate in spontaneous contractions of small intestine. On the other hand, voltage-dependent K+ channels, HERG K+ channels, inward rectifier K+ channels and high conductance Ca2+-activated K+ channels are involved in KCl-stimulated contractions.     

In vitro propagation of the neotropical giant bamboo, Guadua angustifolia Kunth, through axillary shoot proliferation

Guadua angustifolia Kunth was successfully propagated in vitro from axillary buds. Culture initiation, bud sprouting, shoot and plant multiplication, rooting and acclimatization, were evaluated. Best results were obtained using explants from greenhouse-cultivated plants, following a disinfection procedure that comprised the sequential use of an alkaline detergent, a mixture of the fungicide Benomyl and the bactericide Agri-mycin, followed by immersion in sodium hypochlorite (1.5% w/v) for 10 min, and culturing on Murashige and Skoog medium containing 2 ml l^?1 of Plant Preservative Mixture^®. Highest bud sprouting in original explants was observed when 3 mg l^?1 N⁶-benzylaminopurine (BAP) was incorporated into the culture medium. Production of lateral shoots in in vitro growing plants increased with BAP concentration in culture medium, up to 5 mg l^?1, the highest concentration assessed. After six subcultures, clumps of 8–12 axes were obtained, and their division in groups of 3–5 axes allowed multiplication of the plants. Rooting occurred in vitro spontaneously in 100% of the explants that produced lateral shoots. Successful acclimatization of well-rooted clumps of 5–6 axes was achieved in the greenhouse under mist watering in a mixture of soil, sand and rice hulls (1:1:1).