Expression of Mycobacterium leprae HSP65 in tobacco and its effectiveness as an oral treatment in adjuvant-induced arthritis

Transgenic plants are able to express molecules with antigenic properties. In recent years, this has led the pharmaceutical industry to use plants as alternative systems for the production of recombinant proteins. Plant-produced recominant proteins can have important applications in therapeutics, such as in the treatment of rheumatoid arthritis (RA). In this study, the mycobacterial HSP65 protein expressed in tobacco plants was found to be effective as a treatment for adjuvant-induced arthritis (AIA). We cloned the hsp65 gene from Mycobacterium leprae into plasmid pCAMBIA 2301 under the control of the double 35S promoter from cauliflower mosaic virus. Agrobacterium tumefaciens bearing the pChsp65 plasmid was used to transform tobacco plants. Incorporation of the hsp65 gene was confirmed by PCR, reverse transcription-PCR, histochemistry, and western blot analyses in several transgenic lines of tobacco plants. Oral treatment of AIA rats with the HSP65 protein allowed them to recover body weight and joint inflammation was reduced. Our results suggest a synergistic effect between the HSP65 expressed protein and metabolites presents in tobacco plants.

Low protein diet changes the energetic balance and sympathetic activity in brown adipose tissue of growing rats

Objective: The aim of this study was to assess the effects of protein restriction in growing rats. Methods: Rats (approximate weight, 100 g) were maintained with low-protein (LP; 6%) or normo-proteic (control; 17%) diets, and at the end of the 15th day, hormonal and biochemistry parameters and energetic balance were evaluated. Data were analyzed using Student`s t test (with statistical significance set at P <= .05). Results: LP animals were hyperphagic and showed increased energetic gain (24%) and energy expenditure (EE) compared with controls. The increase in EE was followed by increased sympathetic activity in brown adipose tissue, evidenced by increased norepinephrine turnover, suggesting increased thermogenesis. In spite of hyperphagia, protein ingestion in LP animals was lower than that of controls (P < 0.01). The LP diet impaired body growth and caused deep alterations in body chemical composition, with an increase in carcass lipid content (64%) and reductions of protein and water. In LP animals, postprandial glycemia was unchanged, and insulinemia was lower than in controls (P <= .01). Reduction in fasting glycemia without changes in insulinemia also was detected (P < .01), suggesting increased insulin sensitivity. The LP diet caused a 100% increase in serum leptin (P < .01). Conclusions: Protein restriction led to an increase in EE, with probable activation of thermogenesis in brown adipose tissue, evidenced by an increase in catecholamines levels. Despite the higher EE, energetic gain and lipids increased. The high level of leptin associated with hyperphagia led to the supposition that these animals are leptin resistant, and the increase in insulin sensitivity, suggested by the relation between insulin and glycemia in fasting and fed animals, might contribute to lipid accumulation. (C) 2009 Elsevier Inc. All rights reserved.

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.

Mars is close to venus - Female reproductive proteins are expressed in the fat body and reproductive tract of honey bee (Apis mellifera L.) drones

Vitellogenin (Vg) and lipophorin (Lp) are lipoproteins which play important roles in female reproductive physiology of insects. Both are actively taken up by growing oocytes and especially Vg and its receptor are considered as female-specifically expressed. The finding that the fat body of in honey bee (Apis mellifera) drones synthesizes Vg and is present in hemolymph has long been viewed as a curiosity. The recent paradigm change concerning the role played by Vg in honey bee life history, especially social division of labor, has now led us to investigate whether a physiological constellation similar to that seen in female reproduction may also be represented in the male sex. By means of Western blot analysis we could show that both Vg and Lp are present in the reproductive tract of adult drones, including the accessory (mucus) glands, but apparently are not secreted. Furthermore, we analyzed the transcript levels of the genes encoding these proteins (vg and lp), as well as their putative receptors (Amvgr and Amlpr) in fat body and accessory glands. Whereas lp, vg and Amlpr transcript levels decreased with age in both tissues. Amvgr mRNA levels increased with age in fat body. To our knowledge this is the first report that vitellogenin and its receptor are co-expressed in the reproductive system of a male insect. We interpret these findings as a cross-sexual transfer of a social physiological trait, associated with the rewiring of the juvenile hormone/vitellogenin circuitry that occurred in the female sex of honey bees. (C) 2010 Elsevier Ltd. 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.

Experimental model for study of anorectal sphincter musculature by manometry and computerized tomography in piglets

There seems to be controversy on the anorectal sphincter presentation and anatomical division, as well as on its functional representation. Evaluation of the anorectal sphincter musculature has been achieved through several methods, including anorectal manometry and computerized tomography, but to date there is no experimental model allowing a detailed manometric study of this muscle complex. In this work, we have developed such a model, which should enable the manometric and radiographic study of the anatomical features and functional mechanisms of sphincteric injuries, as well as the assessment of drug effects on the anorectal musculature upon incontinence and constipation. Twenty-two piglets (aged 25-30 days, weighing 5-7 kg) were studied by anorectal manometry (rectoanal inhibitory reflex and vector volume) and computerized tomography (anorectal angle and anal canal length). The data obtained for the rectoanal inhibitory reflex, represented here as the average and standard deviation, were the following: relaxation duration = 14.75 +/- 3.62 s, sphincter basal pressure = 41.58 +/- 8.20 mmHg, relaxation index = 87.26 +/- 11.52%, speed of relaxation = 5.90 +/- 2.10 mm/s, and speed of relaxation recovery = 4.03 +/- 1.78 mm/s. As for the vector volume, results were as follows: vector volume = 2692.32 +/- 1298.12 mmHg(2) cm, sphincter length = 11.82 +/- 2.74 mm, high pressure zone length = 5.09 +/- 1.34 mm, maximum pressure = 61.50 +/- 20.58 mmHg, and asymmetry index = 43.50 +/- 10.03%. Radiographic evaluation led to the following results: anal canal length = 9.61 +/- 2.14 mm and anorectal angle = 137.91 +/- 7.75 degrees. The experimental model designed here allows both anorectal manometry and computerized tomography to be carried out in the same way it is performed in human beings, as long as animal sedation is strictly controlled.

Roux-en-Y Bypass Gastroplasty: Markers of Oxidative Stress 6 Months After Surgery

This study examined the effect of weight loss on energy intake, vitamin C, E, beta-carotene (diet/blood), reduced glutathione (GSH), C-reactive protein (CRP), thiobarbituric acid reactive substances (TBARS), catalase, and myeloperoxidase, in patients with Roux-en-Y bypass gastroplasty. Prospective clinical study with control (C) and bariatric (B) groups (n = 20 each). Age was 38.8 +/- 11.1 (C) and 37.8 +/- 11.2 years (B), and body mass indices (BMI) were 22.4 +/- 2.4 and 48.1 +/- 8.7 kg/m(2), respectively. Group C was assessed on a single occasion and B at three time points (basal period and 3 and 6 months after gastroplasty). BMI was decreased at three (38.3 +/- 1.7, P = 0.018) and 6 months after surgery (34.9 +/- 1.7, P < 0.001). Mean weight loss was 20.53 +/- 1.1 after three and 27.96 +/- 1.3 kg after 6 months. Serum vitamin C and beta-carotene (P < 0.01 and P < 0.001, respectively) were increased at 6 months compared to basal. Basal serum vitamin C (P = 0.001) and beta-carotene (P < 0.001) were lower compared to controls. Serum vitamin E corrected for cholesterol and triglycerides was higher in group B at three (P = 0.01) and 6 months (P = 0.001) and lower at basal (P < 0.001) compared to controls. GSH was higher in controls (P < 0.001) compared to basal. Catalase (P = 0.01) and TBARS (P < 0.001) were higher in group B at 6 months. TBARS were higher (P < 0.001) at basal compared to controls. Myeloperoxidase and CRP decreased in group B after three (P = 0.028, P = 0.010) and 6 months (P < 0.001, P = 0.001), respectively. Roux-en-Y bypass gastroplasty led to decreased proinflammatory parameters together with increased nutritional antioxidants, catalase, and TBARS, and decreased GSH 6 months after surgery.

Short-Term Carbohydrate-Restricted Diet for Weight Loss in Severely Obese Women

Weight loss in bariatric pre-surgery period reduces surgical complications, surgery time, blood loss, and length of hospital stay. Carbohydrate-restricted diets have been used as an alternative for weight loss. We tested the efficacy of a low-calorie carbohydrate-restricted diet (RD) for short-term weight loss in women with severe obesity and evaluate its metabolic effects in relation to a conventional low-calorie diet (CD). The subjects received a 1,200-kcal diet with or without carbohydrate restriction for a period of 1 week in the hospital. Nineteen obesity class III women were distributed into two groups: experimental (n = 10) and control (n = 9). The following variables were assessed at the beginning and end of the study: anthropometric measurements, body composition, resting energy expenditure, substrate oxidation, and biochemical tests. Compared with CD, RD led to larger weight loss (2.6 and 4.4 kg, respectively; p = 0.01) and waist circumference reduction (p < 0.01). Among the assessed biochemical indicators, only plasma and urine acetone levels were different (p < 0.01); higher values were found in the experimental group with no symptoms and other diet-related complaints. There was also a significant decrease in triglycerides and carbohydrate oxidation, as well as a significant enhancement in lipid oxidation in the RD group. Short-term RD was more efficient than CD regarding quick weight loss and waist circumference reduction, which may favor gastroplasty. Also, RD did not lead adverse metabolic effects.

Protective Effect of RC-3095, an Antagonist of the Gastrin-Releasing Peptide Receptor, in Experimental Arthritis

Objective. To evaluate the antiinflammatory effects of RC-3095 in 2 experimental models of arthritis, collagen-induced arthritis (CIA) and antigen-induced arthritis (AIA), and to determine the mechanisms of action involved. Methods. RC-3095 was administered daily to mice with CIA and mice with AIA, after induction of disease with methylated bovine serum albumin. Disease incidence and severity were assessed using a clinical index and evaluation of histologic features, respectively. In mice with CIA, gastrin-releasing peptide receptor (GRPR) was detected by immunohistochemical analysis, while in mice with AIA, migration of neutrophils, presence of glycosaminoglycans, and lymphocyte proliferation, determined using the MTT assay, were assessed. Expression of cytokines interleukin-17 (IL-17), IL-1 beta, and tumor necrosis factor alpha (TNF alpha) was evaluated in all mouse knees using enzyme-linked immunosorbent assay. Treg cell production was assessed by flow cytometry in the joints of mice with AIA. Results. In mice with AIA, administration of RC-3095 reduced neutrophil migration, mechanical hypernociception, and proteoglycan loss. These findings were associated with inhibition of the levels of all 3 proinflammatory cytokines, decreased lymphocyte proliferation, and increased Treg cell numbers. In the CIA model, treatment with RC-3095 led to a significant reduction in arthritis clinical scores and the severity of disease determined histologically. Synovial inflammation, synovial hyperplasia, pannus formation, and extensive erosive changes were all dramatically reduced in the arthritic mice treated with RC-3095. Furthermore, arthritic mice treated with RC-3095 showed a significant reduction in the concentrations of IL-17, IL-1 beta, and TNF alpha, and showed a diminished expression of GRPR. Conclusion. These findings suggest that the GRP pathway has a significant role in chronic arthritis, and its inhibition can be explored as a possible therapeutic strategy in rheumatoid arthritis.

IL-4 regulates susceptibility to intestinal inflammation in murine food allergy

Cardoso CR, Provinciatto PR, Godoi DF, Ferreira BR, Teixeira G, Rossi MA, Cunha FQ, Silva JS. IL-4 regulates susceptibility to intestinal inflammation in murine food allergy. Am J Physiol Gastrointest Liver Physiol 296: G593-G600, 2009. First published January 8, 2009; doi:10.1152/ajpgi.90431.2008.-Allergies involve a state of immediate hypersensitivity to antigens, including food proteins. The mechanism underlying the initiation and development of allergic responses involves IL-4 that directly induces the differentiation of committed effector Th2 lymphocytes. Although it is clear that Th2 responses play a pivotal role in the development of allergic responses, it remains unclear which mechanisms are involved in the development of the intestinal damages observed in food allergy. Accordingly, this work aimed to study the role of Th2/IL-4-dependent responses in the development of food allergy and intestinal pathology. C57BL/6 wild-type (WT) and IL-4(-/-) mice were sensitized with peanut proteins, challenged with peanut seeds, and followed for the development of food allergy and intestinal inflammation. Results demonstrated that exposure to peanut seeds led to weight loss in WT but not in IL-4(-/-) mice that preserved gut integrity with no signs of mucosal inflammation. These animals presented increased levels of IgG2a in sera, suggesting a role for allergic antibodies in the pathogenesis of WT animals. Most importantly, results also showed that lack of IL-4 modulated gut mucosal response in food allergy through diminished expression of TNF-alpha mRNA, increased Th1 IFN-gamma, IL-12p40, regulatory cytokines, and Foxp3, demonstrating their relevance in the control of allergic inflammatory processes, especially in the intestine. Finally, this study highlighted some of the complex mechanisms involved in the pathogenesis of allergic responses to food antigens in the gut, thereby providing valuable tools for directing novel therapeutic or preventive strategies to the control of allergic enteropathy.

Modulation of mucosal immunity in a murine model of food-induced intestinal inflammation

Background Hypersensitivity or uncontrolled responses against dietary antigens can lead to inflammatory disorders like food allergy and current models reflect a variety of causes but do not reveal the detailed modulation of gut immunity in response to food antigens after breakdown in mucosal tolerance. Objective To develop and characterize a murine model for food-induced intestinal inflammation and to demonstrate the modulation of gut immune response by dietary allergenic antigens. Methods C57BL/6 mice were sensitized with peanut proteins, challenged with peanut seeds and their sera and gut segments were collected for subsequent analyses. Results Sensitization and challenged with peanut seeds led to alterations in gut architecture with inflammatory response characterized by oedema in lamina propria and cell infiltrate composed mainly by eosinophils, mast cells, phagocytes, natural killer and plasma cells, together with low percentage of gamma delta(+) and CD4(+)CD25(+)Foxp3(+) cells in Peyer`s patches. These animals also presented high levels of specific IgE and IgG1 in sera and modulation of mucosal immunity was mediated by increased expression of GATA-3, IL-4, IL-13 and TNF-alpha in contrast to low IFN-gamma in the gut. Conclusion A murine model for food-induced intestinal inflammation was characterized in which modulation of gut immunity occurs by peanut antigens in consequence of T-helper type 2 (Th2) allergic response and failure of regulatory mechanisms necessary for mucosa homeostasis, resembling food allergy. This work shed some light on the understanding of the pathogenesis of gastrointestinal disorders and intolerance in the gut and supports the development of therapies for food-related enteropathies like food allergy, focusing on gut-specific immune response.

The use of strategic visioning to enhance local tourism planning in periphery communities

Tourism has had a profound impact upon destinations worldwide, and although this impact has been positive for many destinations, there are numerous examples where tourism has adversely impacted upon the environment and social fabric of the destination community (Coccossis 1996; Murphy 1985). The negative impacts of tourism have been attributed, among other things, to inadequate or non-existent planning for development (Gunn 1994; Hall2000). This has led to increased calls for tourism planning to offset some of the negative impacts that tourism can have on the destination community. While a number of approaches have been advocated, a collaborative philosophy, based on the principles of sustainability, is more likely to result in acceptable and successful policies and programmes for tourism destinations (Farrell1986; Jamal & Getz 1995; Maitland 2002; Minca & Getz 1995). Such an approach focuses on cooperation and broader based participation in tourism planning and decision-making between stakeholders to lead to agreement on planning directions and goals, with one of the primary objectives of collaborative arrangements being to develop a strategic vision for a destination (Bramwell & Lane 2000). [Extract from introduction]

Mitochondrial population genomics supports a single pre-Clovis origin with a coastal route for the peopling of the Americas

It is well accepted that the Americas were the last continents reached by modern humans, most likely through Beringia. However, the precise time and mode of the colonization of the New World remain hotly disputed issues. Native American populations exhibit almost exclusively five mitochondrial DNA (mtDNA) haplogroups (A-D and X). Haplogroups A-D are also frequent in Asia, suggesting a northeastern Asian origin of these lineages. However, the differential pattern of distribution and frequency of haplogroup X led some to suggest that it may represent an independent migration to the Americas. Here we show, by using 86 complete mitochondrial genomes, that all Native American haplogroups, including haplogroup X, were part of a single founding population, thereby refuting multiple-migration models. A detailed demographic history of the mtDNA sequences estimated with a Bayesian coalescent method indicates a complex model for the peopling of the Americas, in which the initial differentiation from Asian populations ended with a moderate bottleneck in Beringia during the last glacial maximum (LGM), around similar to 23,000 to similar to 19,000 years ago. Toward the end of the LGM, a strong population expansion started similar to 18,000 and finished similar to 15,000 years ago. These results support a pre-Clovis occupation of the New World, suggesting a rapid settlement of the continent along a Pacific coastal route.

Preimplantation development and expression of Hsp-70 and Bax genes in bovine blastocysts derived from oocytes matured in alpha-MEM supplemented with growth factors and synthetic macromolecules

In vitro culture conditions affect both the maternal and embryonic expression of genes and is likely to alter both oocyte and embryo developmental competence. The search for better and less variable culture conditions simulating those in vivo has led to the development of defined culture media, with lower impact on the molecular reprogramming of oocytes and embryos. We evaluated embryo development and relative abundance (RA) of Hsp-70 and Bax transcripts in bovine blastocysts produced from oocytes matured in a chemically defined IVM system with synthetic polymers. Immature cumulus oocyte complexes (COCs) were matured for 22-24 h in alpha-MEM supplemented with IGF-1, insulin, 0.1% polyvinyl alcohol (PVA), or 0.1% polyvinylpyrrolidone (PVP), but without FSH or LH. The control group consisted of COCs matured it, TCM plus FSH and 10% estrous cow serum. After fertilization. presumptive zygotes were co-cultured with cumulus cells until 224 h post-insemination. Total RNA was isolated from embryo pools, reverse transcribed into cDNA, and subjected to transcript analysis by real-time PCR. Cleavage rate was higher (P < 0.05) for the control group (68.3%) than for the PVA (54.4%) and PVP-40 (58.3%) groups. Nevertheless. there was no difference among the PVA, PVP-40 and control groups in blastocyst or hatching rates. similarly, no difference in relative abundance of Hsp-70 and Bax transcripts was detected in comparison to the control group. We inferred that bovine oocytes can be matured in serum- and gonadotrophin-free medium supplemented with PVA or PVP, enriched with IGF-I and insulin, without altering post-cleavage development and relative abundance of some genes associated with stress and apoptosis. (C) 2009 Elsevier Inc. All rights reserved.

Calpain5 expression is decreased in endometriosis and regulated by HOXA10 in human endometrial cells

Calpains have been implicated in the regulation of apoptosis. Here, we identified Calpain5 as a target of HOXA10 transcriptional regulation in endometrial cells as well as its aberrant regulation in endometriosis. Histologically confirmed biopsies of endometriosis were obtained from 20 women. Eutopic endometrium was collected by endometrial biopsy from 30 controls and from the 20 subjects with endometriosis. First trimester decidual samples were obtained from five subjects at the time of pregnancy termination. Immunohistochemistry was used to identify Calpain5 expression. Calpain5 was expressed in endometrial stromal and glandular cells throughout the menstrual cycle and in decidua. Calpain5 protein expression was decreased in both stromal and glandular cells from women with endometriosis compared with that of fertile controls. Human endometrial stromal and epithelial cell lines were transfected with pcDNA/HOXA10, HOXA10 siRNA or respective controls. Quantitative real-time RT-PCR was performed to determine expression of HOXA10 and Calpain5 in each group. Transfection of HESC cells with an HOXA10 expression construct led to increased Calpain5 expression, whereas transfection with siRNA resulted in decreased expression. In conclusion, Calpain5 expression is regulated by HOXA10. Calpain5 expression was decreased in endometriosis likely as a result of decreased HOXA10 expression. Decreased apoptosis in endometrial cells may promote the development of endometriosis through a pathway involving HOXA10, Calpain5 and caspase.