Microinjection of histamine into the cerebellar vermis impairs emotional memory consolidation in mice

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Quantitative aspects of the migration and evolutive asynchronism of Schistosoma mansoni in mice

Two groups of white mice (Mus musculus) were infected with 65 and 440 cercariae transcutaneously. Migration of Schistosoma mansoni from skin to the lungs and to the portal system thereafter was studied through fitting mathematical equations. Six evolutive stages previously defined were used to determine the asynchronic development of parasites in the portal system. Equations the moment of maximum schistosomula recovery in skin and lungs. In the portal system the equations lead to different days of maximum recovery according to each stage. These differences measure quantitatively the asynchronism of S. mansoni.

Association between polyclonal B cell activation and the presence of autoantibodies in mice infected with Yersinia enterocolitica O:3

Eight-week old conventional female Swiss mice were inoculated intravenously with Yersinia enterocolitica O:3. A second group of normal mice was used as control. Five mice from each group were bled by heart puncture and their spleens were removed for spleen cell collection on the 3rd, 5th, 7th, 10th, 14th and 21st day after infection. Immunoglobulin-secreting spleen cells were detected by the isotype-specific protein A plaque assay. Total immunoglobulin levels were determined in mouse serum by single radial immunodiffusion and the presence of autoantibodies was determined by ELISA. We observed a marked increase in the total number of cells secreting immunoglobulins of all isotypes as early as on the 3rd day post-infection and the peak of secretion occurred on the 7th day. At the peak of the immunoglobulin response, the total number of secreting cells was 19 times higher than that of control mice and most immunoglobulin-secreting cells were of the IgG2a isotype. On the 10th day post-infection, total serum immunoglobul in values were 2 times higher in infected animals when compared to the control group, and continued at this level up to the 21st day post-infection. Serum absorption with viable Y. enterocolitica cells had little effect on antibody levels detected by single radial immunodiffusion. Analysis of serum autoantibody levels revealed that Y. enterocolitica infection induced an increase of anti-myosin and anti-myelin immunoglobulins. The sera did not react with collagen. The present study demonstrates that Y. enterocolitica O:3 infection induces polyclonal activation of murine B cells which is correlated with the activation of some autoreactive lymphocyte clones.

Comparative structural analysis of neuromuscular junctions in mice at different ages

The authors studied the histochemical and ultrastructural modifications that occur in the neuromuscular junctions (NMJ) of fibularis longus muscles of mice with an age range of 3 to 21 months. Twenty-four male and female animals were killed at 3, 5, 14 and 21 months of age: 7 of them at 3 months, 4 of them at 5 month, 9 at 14 months and 4 at 21 months. The fibularis longus muscles were processed and their NMJ examined with the transmission electron microscope. The most relevant changes were associated with the degeneration and retraction of terminal axons, i.e., axons poor in synaptic vesicles with degenerated mitochondria, and exhibiting multivesicular bodies and vacuoles; exposed and widened junctional folds and cytoplasmic processes of Schwann cells located in the synaptic gutter. The presence of lysosomes or lipofuchsin in the juxtajunctional sarcoplasm was also noted. These observations suggest that the phenomena of retraction and budding occur in the NMJ with advancing age, with a predominance of events associated with degeneration, leading to profound changes in NMJ shape.

Anti-ulcerogenic and analgesic activities of the leaves of Wilbrandia ebracteata in mice

Wilbrandia ebracteata (Cogn.) Cogn. is a medicinal plant belonging to the Cucurbitaceae family used popularly as an antiulcer and analgesic medicine. The hydromethanol extract of leaves was investigated to determine its anti-ulcerogenic (ethanol and indomethacin induced gastric damage) and analgesic (writhing and tail-flick tests) activities in mice (efficacy), its acute toxicity (safety), and its phytochemistry (quality control). Oral administration of leaf extract at a dose of 1000 mg/kg body wt. significantly reduced 73.3% of the total area of lesion in ethanol-induced gastric damage, but was inactive in an indomethacin-induced gastric damage test. The hydromethanol extract was also inactive in both analgesic tests. Oral administration of the leaf extract did not produce mortality in mice, while the LD50 value of the roots was 22.10 mg/kg body wt. in female mice and 58.31 mg/kg body wt. in male mice. Leaves of W. ebracteata reacted positively for steroids, flavonols, flavanones, saponins, tannins and xanthones and negative for other compounds, including cucurbitacins. Leaf extract of W. ebracteata was active as an anti-ulcerogenic, probably through increasing gastric defensive factors, and flavonoids might be the main constituent responsible for this activity.

Refining the intestinal motility test in mice to reduce animal stress

A new protocol using 3-h fast animal for intestinal motility test was developed in our laboratory aiming the 3R's concept to reduce the stress of animals. Our results may aid in formulating recommendations that can be included in revised guidelines with regard to fasting time of mice.

Protocol to refine intestinal motility test in mice

We propose a new protocol intended to conform to the 3Rs (replacement, reduction, and refinement) principle, using animals fasted for 3 h to control intestinal motility, which reduced stress in the animals. In this new protocol, mice are deprived of food for a short time (3 h) and are not killed. The mice are observed until evacuation containing charcoal is observed, and the experimental results are based on the charcoal evacuation time. The present study may aid the formulation of recommendations that can be included in revised guidelines relating to the fasting time of mice. This new concept of an intestinal motility test conforms with respectful science.

A new firefly luciferase with bimodal spectrum: Identification of structural determinants of spectral pH-sensitivity in firefly luciferases

Fireflies emit flashes in the green-yellow region of the spectrum for the purpose of sexual attraction. The bioluminescence color is determined by the luciferases. It is well known that the in vitro bioluminescence color of firefly luciferases can be shifted toward the red by lower pH and higher temperature; for this reason they are classified as pH-sensitive luciferases. However, the mechanism and structural origin of pH sensitivity in fireflies remains unknown. Here we report the cloning of a new luciferase from the Brazilian twilight active firefly Macrolampis sp2, which displays an unusual bimodal spectrum. The recombinant luciferase displays a sensitive spectrum with the peak at 569 nm and a shoulder in the red region. Comparison of the bioluminescence spectra of Macrolampis, Photinus and Cratomorphus firefly luciferases shows that the distinct colors are determined by the ratio between green and red emitters under luciferase influence. Comparison of Macrolampis luciferase with the highly similar North American Photinus pyralis luciferase (91%) showed few substitutions potentially involved with the higher spectral sensitivity in Macrolampis luciferase. Site-directed mutagenesis showed that the natural substitution E354N determines the appearance of the shoulder in the red region of Macrolampis luciferase bioluminescence spectrum, helping to identify important interactions and residues involved in the pH-sensing mechanism in firefly luciferases. © 2005 American Society for Photobiology.

Evaluation of Strychnos pseudoquina St. Hil. leaves extract on gastrointestinal activity in mice

Strychnos pseudoquina St. Hil. (Loganiaceae) was investigated for its ability to protect the gastric mucosa against injuries caused by non-steroidal anti-inflammatory drugs (piroxicam) and a necrotizing agent (HCl/EtOH) in mice. The MeOH extract and enriched alkaloidic fraction (EAF) provided significant protection in experimental models wheer used at doses of 250 and 1000 mg/kg. In vivo tests were carried out to evaluate for possible toxic effects and no mortality was observed up to the 5 g/kg dose level. Phytochemical investigation led to the isolation of a new indole alkaloid, which elucidated the observed pharmacological effects. © 2005 Pharmaceutical Society of Japan.

Low ethanol consumption induces enhancement of insulin sensitivity in liver of normal rats

Moderate amounts of alcohol intake have been reported to have a protective effect on the cardiovascular system and this may involve enhanced insulin sensitivity. We established an animal model of increased insulin sensitivity by low ethanol consumption and here we investigated metabolic parameters and molecular mechanisms potentially involved in this phenomenon. For that, Wistar rats have received drinking water either without (control) or with 3% ethanol for four weeks. The effect of ethanol intake on insulin sensitivity was analyzed by insulin resistance index (HOMA-IR), intravenous insulin tolerance test (IVITT) and lipid profile. The role of liver was investigated by the analysis of insulin signaling pathway, GLUT2 gene expression and tissue glycogen content. Rats consuming 3% ethanol showed lower values of HOMA-IR and plasma free fatty acids (FFA) levels and higher hepatic glycogen content and glucose disappearance constant during the IVITT. Neither the phosphorylation of insulin receptor (IR) and insulin receptor substrate-1 (IRS-1), nor its association with phosphatidylinositol-3-kinase (PI3-kinase), was affected by ethanol. However, ethanol consumption enhanced liver IRS-2 and protein kinase B (Akt) phosphorylation (3 times, P < 0.05), which can be involved in the 2-fold increased (P < 0.05) hepatic glycogen content. The GLUT2 protein content was unchanged. Our findings point out that liver plays a role in enhanced insulin sensitivity induced by low ethanol consumption. © 2005 Elsevier Inc. All rights reserved.

Evaluation of antidiarrhoeal effects of Psidium guajava L. (Myrtaceae) aqueous leaf extract in mice

A crude aqueous extract of the leaves of the guava tree, Psidium guajava L. (Myrtaceae), were studied for antidiarrhoeal effects, to place one of its traditional medical uses. The extract induced a decrease in the propulsive movements of the intestinal contents in mice. These findings suggested that an aqueous extract of guava leaves may be used as an effective treatment for non-specific diarrhoea in folk medicine.

Citotoxicity and immune response induced by organopalladium(II) compounds in mice bearing Ehrlich ascites tumour

Cyclometallated palladium(II) complexes are reactive inorganic compounds employed in several biological studies because of their antitumour potential and interaction with immune system. In the present study, the immune and citotoxic response induced by two organopalladated complexes: [{Pd(N,C-dmba)} 2(μ-NCS) 2] (1), [Pd(C-dmba)(NCS)(dppp)] (2) [dmba = N,N′-dimethylbenzylamine, dppp = 1,3-bis(diphenylphosphino)propane] and cisplatin (cis-DDP), as standard, were investigated in mice bearing Ehrlich ascites tumour. The mice were divided into five groups and inoculated with the compounds (1) or (2) or cisplatin, or only vehicle or phosphate-buffered saline (PBS). Many parameters were evaluated, such as tumour cell percentage in the peritoneal exsudate, levels of seric nitric oxide (NO) and tumour necrosis factor-alpha (TNF-α) and increase in life span. Analysis of all data revealed, for compound (2), an activity similar to that presented by cisplatin, resulting in increased life span, lower levels of seric TNF-α and increase in NO production. ©2007 Sociedade Brasileira de Química.

The structural origin and biological function of pH-sensitivity in firefly luciferases

Firefly luciferases are called pH-sensitive because their bioluminescence spectra display a typical red-shift at acidic pH, higher temperatures, and in the presence of heavy metal cations, whereas other beetle luciferases (click beetles and railroadworms) do not, and for this reason they are called pH-insensitive. Despite many studies on firefly luciferases, the origin of pH-sensitivity is far from being understood. This subject is revised in view of recent results. Some substitutions of amino-acid residues influencing pH-sensitivity in firefly luciferases have been identified. Sequence comparison, site-directed mutagenesis and modeling studies have shown a set of residues differing between pH-sensitive and pH-insensitive luciferases which affect bioluminescence colors. Some substitutions dramatically affecting bioluminescence colors in both groups of luciferases are clustered in the loop between residues 223-235 (Photinus pyralis sequence). A network of hydrogen bonds and salt bridges involving the residues N229-S284-E311-R337 was found to be important for affecting bioluminescence colors. It is suggested that these structural elements may affect the benzothiazolyl side of the luciferin-binding site affecting bioluminescence colors. Experimental evidence suggest that the residual red light emission in pH-sensitive luciferases could be a vestige that may have biological importance in some firefly species. Furthermore, the potential utility of pH-sensitivity for intracellular biosensing applications is considered. © The Royal Society of Chemistry and Owner Societies.