Aspidosperma (Apocynaceae) plant cytotoxicity and activity towards malaria parasites. Part I: Aspidosperma nitidum (Benth) used as a remedy to treat fever and malaria in the Amazon

Infusions of Aspidosperma nitidum (Apocynaceae) wood bark are used to treat fever and malaria in the Amazon Region. Several species of this family are known to possess indole alkaloids and other classes of secondary metabolites, whereas terpenoids, an inositol and the indole alkaloids harmane-3 acid and braznitidumine have been described in A. nitidum . In the present study, extracts from the wood bark, leaves and branches of this species were prepared for assays against malaria parasites and cytotoxicity testing using human hepatoma and normal monkey kidney cells. The wood bark extracts were active against Plasmodium falciparum and showed a low cytotoxicity in vitro, whereas the leaf and branch extracts and the pure alkaloid braznitidumine were inactive. A crude methanol extract was subjected to acid-base fractionation aimed at obtaining alkaloid-rich fractions, which were active at low concentrations against P. falciparum and in mice infected with and sensitive Plasmodium berghei parasites. Our data validate the antimalarial usefulness of A. nitidum wood bark, a remedy that can most likely help to control malaria. However, the molecules responsible for this antimalarial activity have not yet been identified. Considering their high selectivity index, the alkaloid-rich fractions from the plant bark might be useful in the development of new antimalarials.     

Physical exercise-induced changes in the core body temperature of mice depend more on ambient temperature than on exercise protocol or intensity

The mechanisms underlying physical exercise-induced hyperthermia may be species specific. Therefore, the present study aimed to investigate the effects of exercise intensity and ambient temperature on the core body temperature (T _core) of running mice, which provide an important experimental model for advancing the understanding of thermal physiology. We evaluated the influence of different protocols (constant- or incremental-speed exercises), treadmill speeds and ambient temperatures (T _a) on the magnitude of exercise-induced hyperthermia. To measure T _core, a telemetric sensor was implanted in the abdominal cavity of male adult Swiss mice under anesthesia. After recovering from the surgery, the animals were familiarized to running on a treadmill and then subjected to the different running protocols and speeds at two T _a: 24 °C or 34 °C. All of the experimental trials resulted in marked increases in T _core. As expected, the higher-temperature environment increased the magnitude of running-induced hyperthermia. For example, during incremental exercise at 34 °C, the maximal T _core achieved was increased by 1.2 °C relative to the value reached at 24 °C. However, at the same T _a, neither treadmill speed nor exercise protocol altered the magnitude of exercise-induced hyperthermia. We conclude that T _core of running mice is influenced greatly by T _a, but not by the exercise protocols or intensities examined in the present report. These findings suggest that the magnitude of hyperthermia in running mice may be regulated centrally, independently of exercise intensity.     

Ab initio simulations of Cu binding sites on the N-terminal region of prion protein

The human prion protein binds Cu²⁺ ions in the octarepeat domain of the N-terminal tail up to full occupancy at pH 7.4. Recent experiments have shown that the HGGG octarepeat subdomain is responsible for holding the metal bound in a square-planar configuration. By using first principle ab initio molecular dynamics simulations of the Car–Parrinello type, the coordination of copper to the binding sites of the prion protein octarepeat region is investigated. Simulations are carried out for a number of structured binding sites. Results for the complexes Cu(HGGGW)(wat), Cu(HGGG), and [Cu(HGGG)]₂ are presented. While the presence of a Trp residue and a water molecule does not seem to affect the nature of the copper coordination, high stability of the bond between copper and the amide nitrogen of deprotonated Gly residues is confirmed in all cases. For the more interesting [Cu(HGGG)]₂ complex, a dynamically entangled arrangement of the two domains with exchange of amide nitrogen bonds between the two copper centers emerges, which is consistent with the short Cu–Cu distance observed in experiments at full copper occupancy.     

Calreticulin expression levels and endoplasmic reticulum during late oogenesis and early embryogenesis of <Emphasis Type="Italic">Rhodnius prolixus</Emphasis> Stahl

This study reports the cloning, expression analysis and localization of calreticulin (CRT) in the endoplasmic reticulum (ER) during late oogenesis and early embryogenesis of the insect Rhodnius prolixus. CRT was cloned and sequenced from cDNA extracted from unfertilized eggs. Real-time PCR showed that CRT expression remains at lower levels during late oogenesis when compared to vitellogenic oocytes or day 0 laid fertilized eggs. Immunofluorescence microscopy showed that this protein is located in the periphery of the egg, in a differential peripheral ooplasm surrounding the yolk-rich internal ooplasm, only identified by transmission electron microscopy (TEM) of thin sections. Using immunogold electron microscopy, the ER ultrastructure (CRT labeled) was identified in the peripheral ooplasm as dispersed lamellae, randomly distributed in the peripheral ooplasm. No massive alterations of ER ultrastructure were found before or right after (30 min) fertilization, but an increase in CRT expression levels and assembly of typical rough ER (parallel cisternae with associated ribosomes) were observed 18–24 h after oviposition. The lack of ER assembly at fertilization and the later formation of rough ER together with the increase in CRT expression levels, suggest that the major functions of ER might be of great importance during the early events of development. The possible involvement of ER in the early steps of embryogenesis will be discussed.     

Dose-Finding in the Development of an LPS-Induced Model of Synovitis in Sheep

Models of transient synovitis that can be controlled with antiinflammatory and analgesic drugs have been used to study pain amelioration. To this end, we aimed to determine the dose of intraarticularly administered E. coli LPS that induced signs of synovitis without systemic signs in clinically healthy male castrated sheep (n = 14). In phase 1, a single dose of LPS (0.5, 1.0, 1.5, or 2.0 ng in a total volume of 0.5 mL) was administered into the right stifle joint. In phase 2, a dose of LPS (1.0 or 2.0 μg) in 0.3 mL was administered to 4 naïve sheep. In phase 3, 4 sheep from phase 1 were inoculated after a 60 d washout period with either 0.5 or 1.0 μg of LPS. During the first 48 h after LPS administration, the following were performed: assessment of clinical parameters; scoring for lameness, pain on limb flexion, and local swelling; and ultrasonography of the joints were performed. The doses tested during phase 1 produced subtle signs. During phase 2, mild to moderate lameness with no evidence of systemic signs occurred at both doses. In phase 3, clinical responses were similar between the 0.5- and 1-µg doses. Signs of swelling were not observed at any time. Therefore, we consider the 0.5-µg to be the most appropriate for this model, because it was the lowest dose tested capable of causing lameness without signs of systemic inflammation in all animals.

As an experimental model for the study of arthropathies, the aseptic administration of small doses of endotoxin in the joint induces mild to moderate inflammation and the development of clinical signs similar to those of the naturally occurring disease.⁶ Some studies have used models of transient synovitis to determine whether the associated pain can be controlled with antiinflammatory and analgesic drugs. The use of an LPS-induced model of synovitis to evaluate the analgesic effect of various therapeutic protocols has mainly been reported for horses.^9,16,27,28 However, sheep are an important model species in biomedical research, particularly in orthopedic studies,^15,20,32 due to their similarity in weight, size, and joint and bone structure with humans, and in cardiovascular^7,11 studies, because they are good models of cardiac anatomy and physiology. Consequently, the development of analgesia protocols for acute pain conditions is greatly needed.Animal experiments are under increasing focus regarding their ethical and legal aspects. In vivo studies are permitted when methods consistent with the 3Rs principals (replacement, refinement, and reduction) are considered and implemented.²⁶ This means that experiments have to be performed without animals when possible (replacement) or with as few animals as possible (reduction) and with as little pain and distress as possible (refinement). In this context, species-specific analgesia is considered an important refinement method applicable to the majority of research.²⁵ However, few studies have been conducted to determine analgesia protocols for different pain conditions in sheep. The standardization of animal pain models is necessary for the reliable evaluation of efficient and different drug protocols.^10,19,24To guide standardization of the dose of E. coli LPS for intraarticular administration, with the aim of developing a pain model for studies of analgesia in sheep, we here assessed the ability of various intraarticular doses of LPS to trigger synovitis. Our hypothesis was that the dose established for use in horses (0.5 ng/joint) would trigger similar effects in sheep.