Cytotoxic effects of crotamine are mediated through lysosomal membrane permeabilization

Crotamine, one of the main toxic components of Crotalus durissus terrificus venom, is a small non-enzymatic basic polypeptide, which causes hind limb paralysis and necrosis of muscle cells. it is well-known that several toxins penetrate into the cytosol through endocytosis, although in many cases the mechanism by which this occurs has not been fully investigated. Recently, using low concentrations of crotamine, we demonstrated the uptake of this toxin into actively proliferative cells via endocytosis, an event that ensues crotamine binding to cell membrane heparan sulfate proteoglycans. Thus, crotamine can be regarded as a cell-penetrating peptide that, additionally, has been shown to be able of delivering some biologically active molecules into various cells. Herein, we investigate one of the mechanisms by which crotamine exerts its cytotoxic effects by following its uptake into highly proliferative cells, as CHO-K1 cells. Crotamine accumulation in the acidic endosomal/lysosomal vesicles was observed within 5 min after treatment of these cells with a cytotoxic concentration of this toxin, a value determined here by classical MTT assay. This accumulation caused disruption of lysosomal vesicles accompanied by the leakage of these vesicles contents into the cytosol. This lysosomal lysis also promoted the release of cysteine cathepsin and an increase of caspase activity in the cytoplasm. This chain of events seems to trigger a cell death process. Overall, our data suggest that lysosomes are the primary targets for crotamine cytotoxicity, a proposal corroborated by the correlation between both the kinetics and concentration-dependence of crotamine accumulation in lysosome compartments and the cytotoxic effects of this protein in CHO-K1 cells. Although crotamine is usually regarded as a myotoxin, we observed that intraperitoneal injection of fluorescently labeled crotamine in living mice led to significant and rapid accumulation of this toxin in the cell cytoplasm of several tissues, suggesting that crotamine cytotoxicity might not be restricted to muscle cells. (C) 2008 Elsevier Ltd. All rights reserved.

Gangliosides Are Important for the Preservation of the Structure and Organization of RBL-2H3 Mast Cells

Gangliosides are known to be important in many biological processes. However, details concerning the exact function of these glycosphingolipids in cell physiology are poorly understood. in this study, the role of gangliosides present on the surface of rodent mast cells in maintaining cell structure was examined using RBL-2H3 mast cells and two mutant cell lines (E5 and D1) deficient in the gangliosides, GM(1) and the alpha-galactosyl derivatives of the ganglioside GD(1b). The two deficient cell lines were morphologically different from each other as well as from the parental RBL-2H3 cells. Actin filaments in RBL-2H3 and E5 cells were under the plasma membrane following the spindle shape of the cells, whereas in D1 cells, they were concentrated in large membrane ruffles. Microtubules in RBL-2H3 and E5 cells radiated from the centrosome and were organized into long, straight bundles. The bundles in D1 cells were thicker and organized circumferentially under the plasma membrane. The endoplasmic reticulum, the Golgi complex, and the secretory granule matrix were also altered in the mutant cell lines. These results suggest that the mast cell-specific alpha-galactosyl derivatives of ganglioside GD(1b) and GM(1) are important in maintaining normal cell morphology. (J Histochern Cytochem 58:83-93, 2010)

Lectins and/or xyloglucans/alginate layers as supports for immobilization of dengue virus particles

Formation of stable thin films of mixed xyloglucan (XG) and alginate (ALG) onto Si/SiO2 wafers was achieved under pH 11.6, 50 mM CaCl2, and at 70 degrees C. XG-ALG films presented mean thickness of (16 +/- 2) nun and globules rich surface, as evidenced by means of ellipsometry and atomic force microscopy (AFM), respectively. The adsorption of two glucose/mannose-binding seed (Canavalia ensiformis and Dioclea altissima) lectins, coded here as ConA and DAlt, onto XG-ALG surfaces took place under pH 5. Under this condition both lectins present positive net charge. ConA and DAIt adsorbed irreversibly onto XG-ALG forming homogenous monolayers similar to(4 +/- 1)nm thick. Lectins adsorption was mainly driven by electrostatic interaction between lectins positively charged residues and carboxylated (negatively charged) ALG groups. Adhesion of four serotypes of dengue virus, DENV (1-4), particles to XG-ALG surfaces were observed by ellipsometry and AFM. The attachment of dengue particles onto XG-ALG films might be mediated by (i) H bonding between E protein (located at virus particle surface) polar residues and hydroxyl groups present on XG-ALG surfaces and (ii) electrostatic interaction between E protein positively charged residues and ALG carboxylic groups. DENV-4 serotype presented the weakest adsorption onto XG-ALG surfaces, indicating that E protein on DENV-4 surface presents net charge (amino acid sequence) different from E proteins of other serotypes. All four DENV particles serotypes adsorbed similarly onto lectin films adsorbed. Nevertheless, the addition of 0.005 mol/L of mannose prevented dengue particles from adsorbing onto lectin films. XG-ALG and lectin layers serve as potential materials for the development of diagnostic methods for dengue. (c) 2008 Elsevier B.V. All rights reserved.

Effects of acute magnesium loading on pulmonary function of stable COPD patients

Background: Magnesium (Mg) use has the potential to promote bronchodilatation and to improve lung function in obstructive diseases. IV administration of Mg during exacerbations of chronic obstructive pulmonary disease (COPD) has led to improved peak flow. This study aimed to investigate the effects of acute IV Mg loading on respiratory parameters of stable COPD patients. Material/Methods: This was a randomized, double-blind, placebo-controlled crossover study. Twenty-two male COPD patients (64 +/- 6 years old, FEV1: 49 +/- 20%) received an IV infusion of 2 g of magnesium sulfate or placebo on two distinct occasions. Spirometry and mouth maximal respiratory pressures were obtained before and 45 minutes after the infusions. Results: Mg use led to significant changes in functional respiratory capacity (-0.48 1,95% CI: -0.96, -0.01), inspiratory capacity (0.21 1,95% CI: 0.04, 0.37). The treatment was also associated with a marginally significant decrease in residual volume (-0.47 1,95% CI: -0.96, 0.02, p=0.06). Conclusions: Acute IV Mg loading in stable COPD patients was associated with a reduction in lung hyperinflation and improvement of respiratory muscle strength. The clinical potential for chronic magnesium supplementation in COPD deserves further investigation.

Evaluation of swallowing in children with vomiting after feeding

Vomiting after feeding is a symptom of gastroesophageal reflux (GER) and of eosinophilic esophagitis (EE), which are considered to be a cause of infant feeding disorder. The objective of the present study was to evaluate swallowing in children with feeding disorder manifested by vomiting after feeding. Using clinical and videofluoroscopic methods we studied the swallowing of 37 children with vomiting after feeding (mean age = 15.4 months), and of 15 healthy children (mean age = 20.5 months). In the videofluoroscopic examination the children swallowed a free volume of milk and 5 ml of mashed banana, both mixed with barium sulfate. We evaluated five swallows of liquid and five swallows of paste. The videofluoroscopic examination was recorded at 60 frames/s. Patients had difficulty during feeding, pneumonia, respiratory distress, otitis, and irritability more frequently than controls. During feeding, children with vomiting, choke were irritable, and refused food more frequently than controls, and during the videofluoroscopic examination the patients had more backward movement of the head than controls for both the liquid and paste boluses. There was no difference in the timing of oral swallowing transit, pharyngeal swallowing transit, or pharyngeal clearance between patients and controls. We conclude that children with vomiting after feeding may have difficulties in accepting feeding, although they have no alteration of oral and pharyngeal phases of swallowing.

Isolation of transcripts overexpressed in the human pathogen Trichophyton rubrum grown in lipid as carbon source

Trichophyton rubrum is the most common etiological agent of human dermatophytosis. Despite the incidence and medical importance of this dermatophyte, little is known about the mechanisms of host invasion and pathogenicity. Host invasion depends on the adaptive cellular responses of the pathogen that allow it to penetrate the skin layers, which are mainly composed of proteins and lipids. In this study, we used suppression subtractive hybridization to identify transcripts over-expressed in T rubrum cultured in lipid as carbon source. Among the subtractive cDNA clones isolated, 85 clones were positively screened by cDNA array dot blotting and were sequenced. The putative proteins encoded by the isolated transcripts showed similarities to fungal proteins involved in metabolism, signaling, defense, and virulence, such as the MDR/ABC transporter, glucan 1,3-beta-glucosidase, chitin synthase B, copper-sulfate-regulated protein, and serine/threonine phosphatase (calcineurin A). These results provide the first molecular insight into the genes differentially expressed during the adaptation of T. rubrum to a lipidic carbon source.

Toothache referred from auriculotemporal neuralgia: case report

P>Aim To present a 52-year-old male patient who complained of intense pain of short duration in the region of the left external ear and in the ipsilateral maxillary second molar that was relieved by blockade of the auriculotemporal nerve in the infratemporal fossa. Summary Extra- and intraoral physical examination revealed a trigger point that reproduced the symptoms upon finger pressure in the ipsilateral auriculotemporal nerve and in the outer auricular pavilion. The patient`s medical history was unremarkable. The maxillary left second molar tooth was not responsive to pulp sensitivity testing and there was no pain upon percussion or palpation of the buccal sulcus. Periapical radiographs revealed a satisfactory root filling in the maxillary left second molar. On the basis of the clinical signs and symptoms, the auriculotemporal was blocked with 0.5 mL 2% lidocaine and 0.5 mL of a suspension containing dexamethasone acetate (8 mg mL(-1)) and dexamethasone disodium sulfate (2 mg mL(-1)), with full remission of pain 6 months later. The diagnosis was auriculotemporal neuralgia. Key learning point Auriculotemporal neuralgia should be considered as a possible cause of nonodontogenic toothache and thus included in the differential diagnoses. The blockade of the auriculotemporal nerve in the infratemporal fossa is diagnostic and therapeutic. It can be achieved with a solution of lidocaine and dexamethasone.

Expression and characterization of HPV-16 L1 capsid protein in Pichia pastoris

Human papillomaviruses (HPVs) are responsible for the most common human sexually transmitted viral infections. Infection with high-risk HPVs, particularly HPV16, is associated with the development of cervical cancer. The papillomavirus L1 major capsid protein, the basis of the currently marketed vaccines, self-assembles into virus-like particles (VLPs). Here, we describe the expression, purification and characterization of recombinant HPV16 L1 produced by a methylotrophic yeast. A codon-optimized HPV16 L1 gene was cloned into a non-integrative expression vector under the regulation of a methanol-inducible promoter and used to transform competent Pichia pastoris cells. Purification of L1 protein from yeast extracts was performed using heparin-sepharose chromatography, followed by a disassembly/reassembly step. VLPs could be assembled from the purified L1 protein, as demonstrated by electron microscopy. The display of conformational epitopes on the VLPs surface was confirmed by hemagglutination and hemagglutination inhibition assays and by immuno-electron microscopy. This study has implications for the development of an alternative platform for the production of a papillomavirus vaccine that could be provided by public health programs, especially in resource-poor areas, where there is a great demand for low-cost vaccines.

Tissue diffusion and retention of metalloproteinases in ascending aortic aneurysms and dissections

Histopathological alterations in human aneurysms and dissections of the thoracic ascending aorta include areas of mucoid degeneration within the medial layer, colocalized with areas of cell disappearance and disruption of extracellular matrix elastic and collagen fibers. We studied the presence of matrix metalloproteinases in relation to their capacity to diffuse through the tissue or to be retained in areas of mucoid degeneration in aneurysms and dissections of the ascending aorta. Ascending aortas from 9 controls, 33 patients with aneurysms, and 14 with acute dissections, all collected at surgery, were analyzed. The morphological aspect was similar whatever the etiology or phenotypic expression of the pathological aortas, involving areas of extracellular matrix breakdown and cell rarefaction associated with mucoid degeneration. Release of proMMP-2, constitutively expressed by smooth muscle cells, was not different between controls and aneurysmal aortas, whereas the aneurysmal aortas released more of the active form. Release of pro and active MMP-9 was also similar between controls and aneurysmal aortas. Immunohistochemical staining of MMP-2 and MMP-9 was weak in both control and pathological aortas. In contrast, released MMP-7 (matrilysin) and MMP-3 (stromelysin-1) could not be detected in conditioned media but were present in tissue extracts with no detectable quantitative difference between controls and pathological aortas. Immunohistochemical staining of MMP-7 and MMP-3 revealed their retention in areas of mucoid degeneration, and semiquantitative evaluation of immunostaining showed more MMP-7 in pathological aortas than in controls. In conclusion, areas of mucoid degeneration, the hallmark of aneurysms, and dissections of thoracic ascending aortas, whatever their etiology, are not inert and can retain specific proteases. (c) 2009 Elsevier Inc. All rights reserved.

Antitumoural effect of an L-amino acid oxidase isolated from Bothrops jararaca snake venom

An L-amino acid oxidase (BjarLAAO-I) from Bothrops jararaca snake venom was highly purified using a stepwise sequential chromatography on Sephadex G-75, Benzamidine Sepharose and Phenyl Sepharose. Purified BjarLAAO-I showed a molecular weight around 60,000 under reducing conditions and about 125,000 in the native form, when analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively. BjarLAAO-I is a homodimeric acidic glycoprotein, pI similar to 5.0, and N-terminal sequence showing close structural homology with other snake venom LAAOs. The purified enzyme catalysed the oxidative deamination of L-amino acids, the most specific substrate being L-Phe. Five amino acids, L-Ser, L-Pro, L-Gly, L-Thr and L-Cys were not oxidized, clearly indicating a significant specificity. BjarLAAO-I significantly inhibited Ehrlich ascites tumour growth and induced an influx of polymorphonuclear cells, as well as spontaneous liberation of H(2)O(2) from peritoneal macrophages. Later, BjarLAAO-I induced mononuclear influx and peritoneal macrophage spreading. Animals treated with BjarLAAO-I showed higher survival time.

Behavioral effects of acute stimulation of kappa-opioid receptors during lactation

The behavioral effects of the K-opioid receptor agonist U69593 were examined in lactating rats. On day 5 of lactation, animals were treated with 0.1 mg/kg of U69593 to determine whether it influences general activity and maternal latencies toward pups. Because little attention has been given to the possibility that pre-mating treatment with morphine may modulate the response to K-opioid receptor stimulation, another group of animals was submitted to the same acute challenge after abrupt withdrawal from repeated treatment with morphine sulfate during the pre-mating period (5 mg/kg on alternate days for a total of five doses). Acute F;opioid stimulation reduced total locomotion, rearing frequency, and time spent self-grooming and increased immobility duration. These K agonist effects were not observed in animals pretreated with morphine. Similarly, latencies to retrieve pups were longer only in animals pretreated with saline and challenged acutely with U69593. None of these effects were observed in morphine sulfate-pretreated animals. The present results suggest that pre-mating repeated exposure to morphine produces a tolerance-like effect on behavioral responses to low-dose K-opioid receptor stimulation in active reproductive females. (c) 2008 Elsevier Inc. All rights reserved.

Calcitonin, sodium alendronate and high intensity laser in the treatment of traumatized teeth: a preliminary study

The aim of this study was to evaluate the influence of erbium:yttrium-aluminum-garnet (Er:YAG) laser compared with traditional treatment on dentin permeability to calcitonin and sodium alendronate. Forty bovine roots were sectioned and divided into eight groups. Groups 1 and 2 (G1/G2) were immersed in saline solution; G1T/G2T were immersed in ethylene diamine tetra-acetic acid plus sodium lauryl ether sulfate (EDTA-T) and sodium hypochlorite (NaOCl); G1I/G2I were irradiated with Er:YAG laser (2.94 mu m, 6 Hz, 40.4 J/cm(2)); G1TI/G2TI were immersed in EDTA-T, NaOCl and subjected to Er:YAG irradiation. After 4 h the radioactivity of the saline solution was measured. Statistical analysis revealed a significant difference (P < 0.05) when the groups treated with EDTA-T and NaOCl followed by Er:YAG laser irradiation were compared with the groups treated with EDTA-T only and with the groups that received no treatment. Er:YAG laser associated with traditional procedures significantly increased the diffusion of calcitonin and sodium alendronate through dentin. All groups showed calcitonin and sodium alendronate diffusion.

Evaluation of the physical and chemical properties of two commercial and three experimental root-end filling materials

Objective. The aim of this study was to evaluate the pH, calcium release, setting time, and solubility of two commercially available mineral trioxide aggregate (MTA) cements (white MTA Angelus and MTA Bio), and of three experimental cements (light-cured MTA, Portland cement with 20% bismuth oxide and 5% calcium sulfate, and an epoxy resin-based cement). Study design. For evaluation of pH and calcium ion release, polyethylene tubes with 1.0 mm internal diameter and 10.0 mm length were filled with the cements and immediately immersed in flasks containing 10 mL deionized water. After 3, 24, 72, and 168 hours, the tubes were removed and the water from the previous container was measured for its pH and calcium content with a pH meter and an atomic absorption spectrophotometer. For analysis of the setting time, Gilmore needles weighing 100 g and 456.5 g were used, in accordance with the American Society for Testing and Materials specification no. C266-03. Solubility of each cement was also tested. Results. All the cements were alkaline and released calcium ions, with a declining trend over time. After 3 hours, Portland cement + bismuth oxide and MTA Bio had the highest pH and light-cured MTA the lowest. After 1 week, MTA Bio had the highest pH and light-cured MTA and epoxy resin-based cement the lowest. Regarding calcium ion release, after 3 hours, Portland cement + bismuth oxide showed the highest release. After 1 week, MTA Bio had the highest. Epoxy resin-based cement and light-cured MTA had the lowest calcium release in all evaluation periods. Regarding setting times, white MTA Angelus and MTA Bio had the shortest, Portland cement + bismuth oxide had an intermediate setting time, and the epoxy resin-based cement had the longest. The materials that showed the lowest solubility values were the epoxy resin-based cement, Portland cement + bismuth oxide, and light-cured MTA. The highest solubility values were presented in white MTA Angelus and MTA Bio. Conclusions. The white MTA Angelus and MTA Bio had the shortest setting times, higher pH and calcium ion release, and the highest solubility. In contrast, the epoxy resin-based cement and light-cured MTA showed lower values of solubility, pH, and calcium ion release. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010; 110: 250-256)

Evaluation of the radiopacity of some commercial and experimental root-end filling materials

Objective. The aim of the present study was to evaluate the radiopacity of 5 root end filling materials (white MTA Angelus, MTA Bio, light-cured MTA, Sealepox RP, and Portland cement clinker with bismuth oxide and calcium sulfate). Method. Five specimens, 10 mm in diameter and 1 mm in thickness according to specification ISO 6876: 2001 were fabricated from each material and radiographed using Insigth occlusal films close to a graduated aluminum step-wedge (2 to 16 mm in thickness). Radiographs were digitized and compared to the aluminum step-wedge. The radiographic density data were converted into millimeters of aluminum (mm Al), using the Digora 1.51 software. Results were evaluated statistically using the analysis of variance (ANOVA) followed by Tukey test. The level of significance was set at 5% (P<.05%). Results. Radiopacity values ranged from 1.21 mm Al (light-cured MTA) to 6.45 mm Al (MTA Angelus). Comparison between materials showed significant difference (P<.05) between MTA Angelus and all other materials, between Sealepox RP and MTA Bio, and between light-cured MTA and Portland cement clinker. Light-cured MTA was significantly less radiopaque than all other materials. No significant difference (P>.05) was found between MTA Bio and Portland cement clinker. Conclusions. All retrograde filling materials evaluated showed greater radiopacity than dentin. All the materials, except light-cured MTA met the minimum radiopacity standards of 3 mm Al recognized by the ISO 6876: 2001 and ADA n.57. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009; 108: e35-e38)

Radiopacity of Portland Cement Associated With Different Radiopacifying Agents

This study evaluated the radiopacity of Portland cement associated with the following radiopacifying agents: bismuth oxide, zinc oxide, lead oxide, bismuth subnitrate, bismuth carbonate, barium sulfate, iodoform, calcium tungstate, and zirconium oxide. A ratio of 20% radiopacifier and 80% white Portland cement by weight was used for analysis. Pure Portland cement and dentin served as controls. Cement/radiopacifier and dentin disc-shaped specimens were fabricated, and radiopacity testing was performed according to the ISO 6876/2001 standard for dental root sealing materials. Using Insight occlusal films, the specimens were radiographed near to a graduated aluminum stepwedge varying from 2 to 16 mm in thickness. The radiographs were digitized and radiopacity compared with the aluminum stepwedge using Digora software (Orion Corporation Soredex, Helsinki, Finland). The radiographic density data were converted into mmAl and analyzed statistically by analysis of variance and Tukey-Kramer test (alpha = 0.05). The radiopacity of pure Portland cement was significantly lower (p < 0.05) than that of dentin, whereas all cement/radiopacifier mixtures were significantly more radiopaque than dentin and Portland cement alone (p < 0.05). Portland cement/bismuth oxide and Portland cement/lead oxide presented the highest radiopacity values and differed significantly from the other materials (p < 0.05), whereas Portland cement/zinc oxide presented the lowest radiopacity values of all mixtures (p < 0.05). All tested substances presented higher radiopacity than that of dentin and may potentially be added to the Portland cement as radiopacifying agents. However, the possible interference of the radiopacifiers with the setting chemistry, biocompatibility, and physical properties of the Portland cement should be further investigated before any clinical recommendation can be done. (J Endod 2009,35:737-740)