mTOR pathway inhibition attenuates skeletal muscle growth induced by stretching

The present study has aimed to verify the influence of calcineurin and mTOR pathways in skeletal muscle longitudinal growth induced by stretching. Male Wistar rats were treated with cyclosporin-A or rapamycin for 10 days. To promote muscle stretching, casts were positioned so as completely to dorsiflex the plantar-flexor muscles at the ankle in one hind limb during the last 4 days of treatment with either cyclosporin-A or rapamycin. Thereafter, we determined soleus length, weight, protein content, and phenotype. In addition, NFATc1, Raptor, S6K1, 4E-BP1, iNOS, and nNOS gene expression in the soleus were determined by real-time polymerase chain reaction. Soleus length, weight, and protein content were significantly reduced by rapamycin treatment in animals submitted to stretching (P<0.05). In contrast, cyclosporin-A treatment did not alter these parameters. In all cyclosporin-A treated groups, there was a significant reduction in NFATc1 expression (P<0.001). Similarly, a significant reduction was noted in Raptor (P<0.001) and S6K1 (P<0.01) expression in all rapamycin-treated groups. No alteration was observed in 4E-BP1 gene expression among rapamycin-treated groups. Stretching increased gene expression of both NOS isoforms in skeletal muscle. Rapamycin treatment did not interfere with NOS gene expression (P<0.05). Cyclosporin-A treatment did not impair muscle growth induced by stretching but instead caused a marked slow-to-fast fiber shift in the soleus; this was attenuated by stretching. The data presented herein indicate that mTOR pathway is involved in skeletal muscle longitudinal growth. We gratefully acknowledge the financial support given by FAPESP.     

New bispyridinium oximes: in vitro and in vivo evaluation of their biological efficiency in soman and tabun poisoning

Improving the efficacy of antidotal treatment of poisonings with nerve agents is still a challenge for the scientific community. This study investigated the interactions of four bispyridinium oximes with human erythrocyte acetylcholinesterase (AChE) and their effects on soman- and tabun-poisoned mice. Oximes HI-6 and TMB-4 were used for comparison. These oximes inhibited AchE with inhibitory potency (IC(50)) ranging from 0.02 to 1.0 mM. The best reactivating potency (%R) was obtained with K074, when AChE was inhibited by tabun. The protective potency (P(50)) of all oximes in human erythrocyte AChE inhibited by soman and tabun could not be determined. In tabun-poisoned mice very good antidotal efficacy was obtained with K027, K048, and K074, which makes them interesting for future investigation. The combination of HI-6 and atropine is the therapy of choice for soman poisoning.     

Effect of the acclimation to high environmental temperature on the activity of hepatic glycogen phosphorylase (a+b and a), liver glycogen content and blood glucose level in rats

(1) Changes in the activity of hepatic glycogen phosphorylase a+b and a (GPh-ase a+b and a), liver glycogen content and blood glucose level during acclimation to moderate high environmental temperature (35±1 °C) were studied. (2) Experiments were carried out on adult fed Wistar rats of both sexes, previously given either short-term (1, 4 and 7 days) or long-term (14, 21, 30 and 60 days) exposure to high environmental temperature. The controls were continuously kept at room temperature (20±2 °C). (3) The results obtained showed that in the period of short-term exposure the liver glycogen content was decreased significantly (after the first and fourth days in male rats and after first day in female rats) and the GPh-ase a activity increased (after first day in male rats and after first, fourth and seventh day in female rats). Long-term exposure caused significant increased liver glycogen content (beginning from the 14th day in male rats and the 21st day in female rats) until the end of the acclimation period (60 days). The elevated activity of GPh-ase a persists after 14th day of exposure only in female rats while there are no significant changes over the rest of the acclimation period in both sexes. There were no significant changes in total GPh-ase activity during the whole period of exposure. Blood glucose level was significantly decreased throughout the whole period of acclimation to high environmental temperature, in both sexes (except in the 1 day exposed groups). (4) The increased activity of hepatic GPh-ase a and decreased glycogen content suggested that the short-term exposure to heat stimulates the glycogenolytical processes. Decreased blood glucose level, and elevated liver glycogen content (r=-0.7467 in male and r=-0.6548 in female rats) suggested that prolonged exposure to high environmental temperature stimulated glycogenogenesis, without changes in the GPh-ase activity.     

Conformational changes of beta-lactoglobulin in sodium bis(2-ethylhexyl) sulfosuccinate reverse micelles. A fluorescence and CD study.

The effect of beta-lactoglobulin encapsulation in sodium bis(2-ethylhexyl) sulfosuccinate reverse micelles on the environment of protein and on Trp was analysed at different water contents (omega0). CD data underlined the distortion of the beta-sheet and a less constrained tertiary structure as the omega0 increased, in agreement with a concomitant red shift and a decrease in the signal intensity obtained in steady-state fluorescence measurements. Fluorescence lifetimes, evaluated by biexponential analysis, were tau1 = 1.28 ns and tau2 = 3.36 ns in neutral water. In reverse micelles, decay-associated spectra indicated the occurrence of important environmental changes associated with omega0. Bimolecular fluorescence quenching by CCl4 and acrylamide was employed to analyse alterations in the accessibility of the two Trp residues in beta-lactoglobulin, induced by changes in omega0. The average bimolecular quenching constant was found not to depend on omega0, confirming the insolubility of this quencher in the aqueous interface, while increases with omega0. The drastic decrease with omega0 of kq, associated with the longest lifetime kq2(CCl4), comparatively to the increase of kq2(acrylamide), emphasizes the location of beta-lactoglobulin in the aqueous interfacial region especially at omega0> or = 10. The fact that (omega0 = 30) > kq2(acrylamide) (water) also confirms the important conformational changes of encapsulated beta-lactoglobulin.     

Intralesional uridine-5′-triphosphate (UTP) treatment induced resistance to <Emphasis Type="Italic">Leishmania amazonensis</Emphasis> infection by boosting Th<Subscript>1</Subscript> immune responses and reactive oxygen species production

Leishmania amazonensis is the etiologic agent of cutaneous leishmaniasis, an immune-driven disease causing a range of clinical symptoms. Infections caused by L. amazonensis suppress the activation and function of immune cells, including macrophages, dendritic cells, and CD4⁺ T cells. In this study, we analyzed the course of infection as well as the leishmanicidal effect of intralesional UTP treatment in L. amazonensis-infected BALB/c mice. We found that UTP treatment reduced the parasitic load in both footpad and lymph node sites of infection. UTP also boosted Th1 immune responses, increasing CD4⁺ T cell recruitment and production of IFN-γ, IL-1β, IL-12, and TNF-α. In addition, the role of UTP during innate immune response against L. amazonensis was evaluated using the air pouch model. We observed that UTP augmented neutrophil chemoattraction and activated microbicidal mechanisms, including ROS production. In conclusion, our data suggested an important role for this physiological nucleotide in controlling L. amazonensis infection, and its possible use as a therapeutic agent for shifting immune responses to Th1 and increasing host resistance against L. amazonensis infection.     

Peripheral blood fibrocytes: new information to explain?the dynamics of Leishmania infection

Fibrocytes are important for understanding the progression of many diseases becausethey are present in areas where pathogenic lesions are generated. However, themorphology of fibrocytes and their interactions with parasites are poorly understood.In this study, we examined the morphology of peripheral blood fibrocytes and theirinteractions with Leishmania (L.) amazonensis . Throughultrastructural analysis, we describe the details of fibrocyte morphology and howfibrocytes rapidly internalise Leishmania promastigotes. Theparasites differentiated into amastigotes after 2 h in phagolysosomes and theinfection was completely resolved after 72 h. Early in the infection, we foundincreased nitric oxide production and large lysosomes with electron-dense material.These factors may regulate the proliferation and death of the parasites. Becausefibrocytes are present at the infection site and are directly involved in developingcutaneous leishmaniasis, they are targets for effective, non-toxic cell-basedtherapies that control and treat leishmaniasis.     

Detection of diarrheagenic Escherichia coli from children with and without diarrhea in Salvador, Bahia, Brazil

We identified different diarrheagenic (DEC) Escherichia coli pathotypes isolated from 1,207 children with and without acute endemic diarrhea in Salvador, Bahia, Brazil collected as part of a case-control study. Since the identification of DEC cannot be based on only biochemical and culture criteria, we used a multiplex polymerase chain reaction developed by combining five specific primer pairs for Enteropathogenic Escherichia coli (EPEC), Shiga toxin-producing E. coli/ Enterohaemorrhagic E. coli (STEC/EHEC), Enterotoxigenic E. coli (ETEC) and Enteroaggregative E. coli (EAEC) to detect these pathotypes simultaneously in a single-step reaction. In order to distinguish typical and atypical EPEC strains, these were tested for the presence of EAF plasmid. The prevalence of diarrheagenic E. coli in this sample of a global case-control study was 25.4% (259 patients) and 18.7% (35 patients) in the diarrhea group (1,020 patients) and the control group (187 patients), respectively. The most frequently isolated pathotype was EAEC (10.7%), followed by atypical EPEC (9.4%), ETEC (3.7%), and STEC (0.6%). Typical EPEC was detected only in one sample. The prevalence of the pathotypes studied in children with diarrhea was not significantly different from that in children without diarrhea.     

Ascorbate uptake is decreased in the hippocampus of ageing rats

Ascorbate, an intracellular antioxidant, has been considered critical for neuronal protection against oxidant stress, which is supported especially by in vitro studies. Besides, it has been demonstrated an age-related decrease in brain ascorbate levels. The aims of the present study were to investigate ascorbate uptake in hippocampal slices from old Wistar rats, as well as its neuroprotective effects in in vitro and in vivo assays. Hippocampal slices from male Wistar rats aged 4, 11 and 24 months were incubated with radiolabeled ascorbate and incorporated radioactivity was measured. Hippocampal slices from rats were incubated with different concentrations of ascorbate and submitted to H(2)O(2)-induced injury, cellular damage and S100B protein levels were evaluated. The effect of chronic administration of ascorbate on cellular oxidative state and astrocyte biochemical parameters in the hippocampus from 18-months-old Wistar rats was also studied. The ascorbate uptake was decreased in hippocampal slices from old-aged rats, while supplementation with ascorbate (2 weeks) did not modify any tested oxidative status in the hippocampus and the incubation was unable to protect hippocampal slices submitted to oxidative damage (H(2)O(2)) from old rats. Our data suggest that the decline of ascorbate uptake might be involved in the brain greater susceptibility to oxidative damage with advancing age and both in vitro and vivo assays suggest that ascorbate supplementation did not protect hippocampal cells.