Differential Toxicity of Disperse Red 1 and Disperse Red 13 in the Ames Test, HepG2 Cytotoxicity Assay, and Daphnia Acute Toxicity Test

Azo dyes are of environmental concern due to their degradation products, widespread use, and low-removal rate during conventional treatment. Their toxic properties are related to the nature and position of the substituents with respect to the aromatic rings and amino nitrogen atom. The dyes Disperse Red 1 and Disperse Red 13 were tested for Salmonella mutagenicity, cell viability by annexin V, and propidium iodide in HepG2 and by aquatic toxicity assays using daphnids. Both dyes tested positive in the Salmonella assay, and the suggestion was made that these compounds induce mainly frame-shift mutations and that the enzymes nitroreductase and O-acetyltransferase play an important role in the observed effect. In addition, it was shown that the presence of the chlorine substituent in Disperse Red 13 decreased the mutagenicity about 14 times when compared with Disperse Red 1, which shows the same structure as Disperse Red 13, but without the chlorine substituent. The presence of this substituent did not cause cytotoxicity in HepG2 cells, but toxicity to the water flea Daphnia similis increased in the presence of the chlorine substituent. These data suggest that the insertion of a chlorine substituent could be an alternative in the design of dyes with low-mutagenic potency, although the ecotoxicity should be carefully evaluated. (C) 2010 Wiley Periodicals, Inc. Environ Toxicol 26: 489-497, 2011.

Lipid microspheres loaded with antigenic membrane proteins of the Leishmania amazonensis as a potential biotechnology application

Lipid microspheres (LM) are excellent drug delivery or vaccines adjuvant systems and are relatively stable. The aim of this work is to develop and characterize a system that is able to encapsulate and present antigenic membrane proteins from Leishmania amazonensis. Membrane proteins are important for vaccine`s formulation because these proteins come in contact with the host cell first, triggering the cell mediated immune response. This is a useful tool to avoid or inactivate the parasite invasion. The LM are constituted by soybean oil (SO), dipalmitoylphosphatidilcholine (DPPC), cholesterol and solubilized protein extract (SPE). The particles formed presented an average diameter of 200 run, low polydispersion and good stability for a period of 30 days, according to dynamic light scattering assays. Isopycnic density gradient centrifugation of LM-protein showed that proteins and lipids floated in the sucrose gradient (5-50%w/v) suggesting that the LM-protein preparation was homogeneous and that the proteins are interacting with the system. The results show that 85% of SPE proteins were encapsulated in the LM. Studies of cellular viability of murine peritoneal macrophages show that our system does not present cytotoxic effect for the macrophages and still stimulates their NO production (which makes its application as a vaccine adjuvant possible). LM-protein loaded with antigenic membrane proteins from L. amazonensis seems to be a promising vaccine system for immunization against leishmaniasis. (C) 2009 Elsevier Inc. All rights reserved.

Monoamine oxidase inhibitory activities of indolylalkaloid toxins from the venom of the colonial spider Parawixia bistriata: Functional characterization of PwTX-I

Colonial spiders evolved a differential prey-capture behaviour in concert with their venom chemistry, which may be a source of novel drugs. Some highly active tetrahydro-beta-carboline (TH beta C) toxins were recently isolated from the venom of the colonial spider Parawixia bistriata; the spiders use these toxins as part of their chemical arsenal to kill and/or paralyze preys. The major TH beta C compound isolated from this venom was identified as 6-hydroxytrypargine, also known as PwTX-I. Most natural compounds of animal origin occur in low abundance, and the natural abundance of PwTX-I is insufficient for complete functional characterization. Thus, PwTx-I was synthesized using a Pictet-Spengler condensation strategy, and the stereoisomers of the synthetic toxin were separated by chiral chromatography. The fraction of venom containing a mixture of three natural TH beta C toxins and enantiomers of PwTx-I were analyzed for inhibition of monoamine oxidase (MAO)-A and -B and for toxicity to insects. We reveal that the mixture of the natural TH beta C toxins, as well as the enantiomers of PwTx-I, were non-competitive inhibitors of MAO-A and MAO-B and caused potent paralysis of honeybees. The (-)-PwTX-I enantiomer is 2-fold more potent than the (+)-PwTX-I enantiomer in the assays performed. (C) 2009 Elsevier Ltd. All rights reserved.

Hemolymph ionic regulation and adjustments in gill (Na(+), K(+))-ATPase activity during salinity acclimation in the swimming crab Callinectes ornatus (Decapoda, Brachyura)

We evaluate hemolymph osmotic and ionic regulatory abilities and characterize a posterior gill microsomal (Na(+), K(+))-ATPase from the marine swimming crab, Callinectes ornatus, acclimated to 21 parts per thousand or 33 parts per thousand salinity. C ornatus is isosmotic after acclimation to 21 parts per thousand but is hyposmotic at 33 parts per thousand salinity; hemolymph ions do not recover initial levels on acclimation to 21 parts per thousand salinity but are anisoionic compared to ambient concentrations, revealing modest regulatory ability. NH(4)(+) modulates enzyme affinity for K(+), which increases 187-fold in crabs acclimated to 33%. salinity. The (Na(+), K(+))-ATPase redistributes into membrane fractions of different densities, suggesting that altered membrane composition results from salinity acclimation. ATP was hydrolyzed at maximum rates of 182.6 +/- 7.1 nmol Pi min(-1) mg(-1) (21 parts per thousand) and 76.2 +/- 3.5 nmol Pi min(-1) mg(-1) (33 parts per thousand), with little change in K(M) values (approximate to 50 mu mol L(-1)). K(+) together with NH(4)(+) synergistically stimulated activity to maximum rates of approximate to 240 nmol Pi min(-1) mg(-1). K, values for ouabain inhibition (approximate to 110 mu mol L(-1)) decreased to 44.9 +/- 1.0 mu mol L(-1) (21 parts per thousand) and 28.8 +/- 1.3 mu mol L(-1) (33 parts per thousand) in the presence of both K(+) and NH(4)(+). Assays employing various inhibitors suggest the presence of mitochondrial F(0)F(1)- and K(+)- and V-ATPase activities in the gill microsomes. (C) 2009 Elsevier Inc. All rights reserved.

Na(+), K(+)-ATPase activity in gill microsomes from the blue crab, Callinectes danae, acclimated to low salinity: Novel perspectives on ammonia excretion

This investigation provides an extensive characterization of the modulation by ATP, Mg(2+), Na(+), K(+) and NH(4)(+) of a gill microsomal (Na(+),K(+))-ATPase from Callinectes danae acclimated to 15 parts per thousand salinity. Novel findings are the lack of high-affinity ATP-binding sites and a 10-fold increase in enzyme affinity for K(+) modulated by NH4+, discussed regarding NH4+ excretion in benthic marine crabs. The (Na(+),K(+))-ATPase hydrolyzed ATP at a maximum rate of 298.7 +/- 16.7 nmol Pi min(-1) mg(-1) and K(0.5) = 174.2 +/- 9.8 mmol L(-1) obeying cooperative kinetics (n(H) = 1.2). Stimulation by sodium (V = 308.9 +/- 15.7 nmol Pi min(-1) mg(-1), K(0.5) = 7.8 +/- 0.4 mmol L(-1)), magnesium (299.2 +/- 14.1 nmol Pi min(-1) mg(-1), K(0.5) = 767.3 +/- 36.1 mmol L(-1)), potassium (300.6 +/- 153 nmol Pi min(-1) mg(-1), K(0.5) = 1.6 +/- 0.08 mmol L(-1)) and ammonium (V = 345.1 +/- 19.0 nmol Pi min(-1) mg(-1), K(0.5) = 6.0 +/- 0.3 mmol L(-1)) ions showed site-site interactions. Ouabain inhibited (Na(+),K(+))-ATPase activity with K(1) = 45.1 +/- 2.5 mu mol L(-1), although affinity for the inhibitor increased (K(1) = 22.7 +/- 1.1 mu mol L(-1)) in 50 mmol L(-1) NH(4)(+). Inhibition assays using ouabain plus oligomycin or ethacrynic acid suggest mitochondrial F(0)F(1)- and K(+)-ATPase activities, respectively. Ammonium and potassium ions synergistically stimulated specific activity up to 72%, inferring that these ions bind to different sites on the enzyme molecule, each modulating stimulation by the other. (C) 2009 Elsevier Inc. All rights reserved.

Root Secretion of Defense-related Proteins Is Development-dependent and Correlated with Flowering Time

Proteins found in the root exudates are thought to play a role in the interactions between plants and soil organisms. To gain a better understanding of protein secretion by roots, we conducted a systematic proteomic analysis of the root exudates of Arabidopsis thaliana at different plant developmental stages. In total, we identified 111 proteins secreted by roots, the majority of which were exuded constitutively during all stages of development. However, defense-related proteins such as chitinases, glucanases, myrosinases, and others showed enhanced secretion during flowering. Defense-impaired mutants npr1-1 and NahG showed lower levels of secretion of defense proteins at flowering compared with the wild type. The flowering-defective mutants fca-1, stm-4, and co-1 showed almost undetectable levels of defense proteins in their root exudates at similar time points. In contrast, root secretions of defense-enhanced cpr5-2 mutants showed higher levels of defense proteins. The proteomics data were positively correlated with enzymatic activity assays for defense proteins and with in silico gene expression analysis of genes specifically expressed in roots of Arabidopsis. In conclusion, our results show a clear correlation between defense-related proteins secreted by roots and flowering time.

Interferon-gamma is required for killing keratinocytes in vitro and in vivo

Epithelial malignancies are common in immunosuppressed individuals and the general population. However the mechanisms by which the adaptive immune system can eliminate immunogenic epithelial cells remain undefined. The aim of this project was to determine the effector molecules required for induction of apoptosis in murine epidermal keratinocytes (MEKs) in vitro and in vivo. HPV16E7-specific CTL lines and T cell receptor transgenic (E7TCRtg) effector cells were obtained from wild type (wt)-C57 and syngeneic mice rendered functionally inactive for perforin (Pfp), interferon-g (IFN-g) or FasL. CTLs or E7TCRtg spleen cells were co-cultured with primary MEKs in vitro or transferred into skin graft recipients. Inhibition of colony formation and skin graft rejection were used as indicators of T cell:KC interaction. Wt E7-specific CTLs and CTLs deficient in perforin, FasL or IFN-g produced mean reductions in colony formation of 67% (62.4–71.3%), 72% (71.1–72%), 76% (73–78%) and 21.5% (14– 34%) respectively. Wt, perforin deficient or FasL deficient CTLs all induced rejection of skin grafts (wt: 6/12; Pfp: 9/15; FasL: 3/13 survival). Transfer and immunisation of wt E7TCRtg spleen cells induces rejection of 50% of grafts (4/8 survival). In contrast, perforin or IFN-g deficient E7TCRtg failed to induce graft rejection (5/6; 4/4 survival). FasL deficient E7TCRtg induced nonspecific rejection of grafts (E7- 2/6 survival; C57- 4/7 survival). Therefore IFN-g production by CTL is necessary and sufficient in vitro and in vivo to kill epithelial cells which express a nonself antigen. Assessment of immunotherapies directed against epithelial tissues may be more effectively achieved by assaying the amount of IFN-g production by CD8 T cells, and the number and affinity of those cells, in conjunction with quantitation of perforin mediated effects in short term assays.

The role of CD4 T help in multiple vaccinations when using minimal CD8 T cell epitope peptides

Background: The fact that some cancers and viral infections can be controlled by effector CD8 T cells led to the possibility of utilising minimal CD8 T cell epitope peptides as vaccines. However using minimal CD8 T cell epitope peptide immunisations and a tumour protection model in mice, we have previously shown that functional memory CD8 T cells are not generated unless CD4 T help is provided at the time of CD8 T cell priming. Short-lived effector cells nevertheless are generated in the absence of T help. Aim: To determine the role of CD4 T help in multiple immunisations. Method: Minimal CD8 T cell peptides of HPV16 E7 protein and Ovalbumin were used (with adjuvants Quil-A or IFA) as immunogens in C57BL mice. The presence of effector CD8 T cells were determined by tumour protection assays and was quantified by IFN-gamma ELISPOT assays. Results: In the present study we show that unless T help is provided at the time CD8 T cells are primed, no CD8 effector cells are generated when boosted with the vaccine again in the absence of T help. Our results further show that this failure could be prevented by the inclusion of a T helper peptide during the primary or booster immunisations.

Search for Chronic Beryllium Disease Among Sarcoidosis Patients in Ontario, Canada

Chronic beryllium disease (CBD) is clinically similar to other granulomatous diseases such as sarcoidosis. It is often misdiagnosed if a thorough occupational history is not taken. When appropriate, a beryllium lymphocyte proliferation tests (BeLPT) need to be performed. We aimed to search for CBD among currently diagnosed pulmonary sarcoidosis patients and to identify the occupations and exposures in Ontario leading to CBD. Questionnaire items included work history and details of possible exposure to beryllium. Participants who provided a history of previous work with metals underwent BeLPTs and an ELISPOT on the basis of having a higher pretest probability of CBD. Among 121 sarcoid patients enrolled, 87 (72%) reported no known previous metal dust or fume exposure, while 34 (28%) had metal exposure, including 17 (14%) with beryllium exposure at work or home. However, none of these 34 who underwent testing had positive test results. Self-reported exposure to beryllium or metals was relatively common in these patients with clinical sarcoidosis, but CBD was not confirmed using blood assays in this population.

Fluoxetine effect on kidney water reabsorption

Background. The pathogenesis of hyponatraemia caused by fluoxetine (Fx) use in the treatment of depression is not well understood. It has been attributed to a SIADH, although ADH-enhanced plasma level has not yet been demonstrated in all the cases reported in humans. This experiment aimed at investigating the effect of fluoxetine on the kidney and more specifically in the inner medullary collecting duct (IMCD). Methods. ( 1) In vivo study: ( a) 10 rats were injected daily i. p. with 10 mg/kg fluoxetine doses. After 10 days, rats were sacrificed and blood and kidneys were collected. (b) Immunoblotting studies for AQP2 protein expression in the IMCD from injected rats and in IMCD tubules suspension from 10 normal rats incubated with 10(-7) M fluoxetine. ( 2) In vitro microperfusion study: The osmotic water permeability (P-f, mu m/s) was determined in normal rats IMCD (n = 6), isolated and perfused by the standard methods. Results. In vivo study: ( a) Injected rats with fluoxetine lost about 12% body weight; Na+ plasma level decreased from 139.3 +/- 0.78 mEq/1 to 134.9 +/- 0.5 mEq/1 ( p < 0.01) and K+ and ADH plasma levels remained unchanged. ( b) Immunoblotting densitometric analysis of the assays showed an increase in AQP2 protein abundance of about 40%, both in IMCDs from injected rats [ control period (cont) 99.6 +/- 5.2 versus Fx 145.6 +/- 16.9, p < 0.05] and in tubule suspension incubated with fluoxetine ( cont 100.0 +/- 3.5 versus 143.0 +/- 2.0, p < 0.01). In vitro microperfusion study fluoxetine increased Pf in the IMCD in the absence of ADH from the cont 7.24 +/- 2.07 to Fx 15.77 +/- 3.25 ( p < 0.01). Conclusion. After fluoxetine use, the weight and plasma Na+ level decreased, and the K+ and ADH plasma levels remained unchanged, whereas the AQP2 protein abundance and water absorption in the IMCD increased, leading us to conclude that the direct effect of fluoxetine in the IMCD could explain at least in part, the hyponatraemia found sometime after this drug use in humans.

Binge eating symptoms, diet composition and metabolic characteristics of obese children and adolescents

This study aimed to determine the occurrence of symptoms of binge eating (BE) among children and adolescents seeking treatment for their obesity, as well as to evaluate their diet composition and metabolic characteristics. The Binge Eating scale (BES) was answered by 128 children and adolescents (10.77 +/- 2.04 years, BMI 29.15 +/- 4.98 kg/m(2), BMI Z score 2.28 +/- 0.46, 53.91% pubescent), who were classified into two subgroups-binge eaters (score greater than or equal to IS points) and non-binge eaters (score lower than 18 points). Anthropometric data, body composition and Tanner stages were collected and dietary evaluation conducted. Blood pressure was determined, and glucose, lipid profile and insulin assays were performed. insulin resistance was determined using HOMA-IR. BE symptoms were present in 39.06% of patients. Carbohydrate intake in diet composition was significantly higher among binge eaters. Children with BE did not demonstrate significant dissimilar metabolic characteristics when compared to their counterparts without BE. Therefore, BE seems to be a prevalent problem among children and adolescents seeking help for their obesity. When associated with obesity, this eating behaviour can influence macronutrient consumption through increased carbohydrate intake. Further research would be valuable to verify the reproducibility of these findings. (c) 2007 Elsevier Ltd. All rights reserved.

Leishmania (Viannia) braziliensis identification by PCR in the state of Para, Brazil

The incidence of cutaneous leishmaniasis (CL) is increasing and there is limited surveillance of Leishmania species throughout the world. We identified the species associated with CL in a region of Amazonia, an area recognized for its Leishmania species variability. Clinical findings were analyzed and correlated with the species identified in 93 patients. PCR assays were based on small subunit ribosomal DNA (SSU-rDNA) and G6PD, and were performed in a laboratory located 3,500 km away. Leishmania (V.) braziliensis was identified in 53 patients (57%). The other 40 patients (43%) carried a different species (including six cases of L (L) amazonensis). Molecular methods can be employed, using special media, to allow transport to distant laboratories. L (V.) braziliensis is the most common species in the area of Para. The location of ulcers can suggest CL species (C) 2010 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

The role of vaccines in the control of STDs: HPV vaccines

Prophylactic vaccines for genital human papillomavirus (HPV) infection have been shown to be feasible in animal models, and suitable vaccine material based on virus-like particles can be produced in bulk at reasonable cost. Initiation of phase III clinical trials will follow definition of trial outcome measures through further epidemiological studies, and development-of assays of host protective immunity. Vaccines could in principle eliminate HPV-related disease, as the human race is the only natural host for the relevant papillomaviruses (PVs). Therapeutic vaccines for genital HPV infection are also possible, but have not yet been demonstrated as feasible in practice because the choice of vaccine antigens is difficult, the method of their optimal delivery is uncertain, and the nature of the relevant antiviral immunity is unknown. PV species specificity will require trials to be conducted in man, which will slow definition of an ideal vaccine.

Protein disulfide isomerase redox-dependent association with p47(phox): evidence for an organizer role in leukocyte NADPH oxidase activation

Mechanisms of leukocyte NADPH oxidase regulation remain actively investigated. We showed previously that vascular and macrophage oxidase complexes are regulated by the associated redox chaperone PDI. Here, we investigated the occurrence and possible underlying mechanisms of PDI-mediated regulation of neutrophil NADPH oxidase. In a semirecombinant cell-free system, PDI inhibitors scrRNase (100 mu g/mL) or bacitracin (1 mM) near totally suppressed superoxide generation. Exogenously incubated, oxidized PDI increased (by similar to 40%), whereas PDIred diminished (by similar to 60%) superoxide generation. No change occurred after incubation with PDI serine-mutated in all four redox cysteines. Moreover, a mimetic CxxC PDI inhibited superoxide production by similar to 70%. Thus, oxidized PDI supports, whereas reduced PDI down-regulates, intrinsic membrane NADPH oxidase complex activity. In whole neutrophils, immunoprecipitation and colocalization experiments demonstrated PDI association with membrane complex subunits and prominent thiol-mediated interaction with p47(phox) in the cytosol fraction. Upon PMA stimulation, PDI was mobilized from azurophilic granules to cytosol but did not further accumulate in membranes, contrarily to p47(phox). PDI-p47(phox) association in cytosol increased concomitantly to opposite redox switches of both proteins; there was marked reductive shift of cytosol PDI and maintainance of predominantly oxidized PDI in the membrane. Pulldown assays further indicated predominant association between PDIred and p47(phox) in cytosol. Incubation of purified PDI (> 80% reduced) and p47(phox) in vitro promoted their arachidonate-dependent association. Such PDI behavior is consistent with a novel cytosolic regulatory loop for oxidase complex (re) cycling. Altogether, PDI seems to exhibit a supportive effect on NADPH oxidase activity by acting as a redox-dependent enzyme complex organizer. J. Leukoc. Biol. 90: 799-810; 2011.

Role of galectin-1 in the olfactory nervous system

The olfactory nervous system is responsible for the detection of odors. Primary sensory olfactory neurons are located in a neuroepithelial sheet lining the nasal cavity. The axons from these neurons converge on to discrete loci or glomeruli in the olfactory bulb. Each glomerulus consists of the termination of thousands of primary axons on the dendrites of second-order olfactory neurons. What are the molecular mechanisms which guide growing olfactory axons to select sites in the olfactory bulb? We have shown that subpopulations of these axons differentially express cell surface carbohydrates and that these different subpopulations target and terminate in particular regions of the olfactory bulb. Interestingly, the olfactory neurons and glial components in the olfactory pathway between the nose and brain express galectin-1. By using in vitro assays of neurite outgrowth we found that both galectin-1 and it's ligands were capable of specifically stimulating neurite elongation. Examination of the olfactory system in galectin-1 null mutants revealed that a subpopulation of axons failed to navigate to their target site in the olfactory bulb. This is the first phenotypic effect observed in galectin-1 null mutants and indicates that galectin-1 has a role in the growth and/or guidance of a subpopulation of axons in the olfactory system during development.