Unraveling the Na,K-ATPase alpha(4) Subunit Assembling Induced by Large Amounts of C(12)E(8) by Means of Small-Angle X-ray Scattering

In the current work, we studied the effect of the nonionic detergent dodecyloctaethyleneglycol, C(12)E(8), on the structure and oligomeric form of the Na,K-ATPase membrane enzyme (sodium-potassium pump) in aqueous suspension, by means of small-angle X-ray scattering (SAXS). Samples composed of 2 mg/mL of Na,K-ATPase, extracted from rabbit kidney medulla, in the presence of a small amount of C(12)E(8) (0.005 mg/mL) and in larger concentrations ranging from 2.7 to 27 mg/mL did not present catalytic activity. Under this condition, an oligomerization of the alpha subunits is expected. SAXS data were analyzed by means of a global fitting procedure supposing that the scattering is due to two independent contributions: one coming from the enzyme and the other one from C(12)E(8) micelles. In the small detergent content (0.005 mg/mL), the SAXS results evidenced that Na,K-ATPase is associated into aggregates larger than (alpha beta)(2) form. When 2.7 mg/mL of C(12)E(8) is added, the data analysis revealed the presence of alpha(4) aggregates in the solution and some free micelles. Increasing the detergent amount up to 27 mg/mL does not disturb the alpha(4) aggregate: just more micelles of the same size and shape are proportionally formed in solution. We believe that our results shed light on a better understanding of how nonionic detergents induce subunit dissociation and reassembling to minimize the exposure of hydrophobic residues to the aqueous solvent.

Aerobic exercise training improves Ca(2+) handling and redox status of skeletal muscle in mice

Exercise training is known to promote relevant changes in the properties of skeletal muscle contractility toward powerful fibers. However, there are few studies showing the effect of a well-established exercise training protocol on Ca(2+) handling and redox status in skeletal muscles with different fiber-type compositions. We have previously standardized a valid and reliable protocol to improve endurance exercise capacity in mice based on maximal lactate steady-state workload (MLSSw). The aim of this study was to investigate the effect of exercise training, performed at MLSSw, on the skeletal muscle Ca(2+) handling-related protein levels and cellular redox status in soleus and plantaris. Male C57BL/6J mice performed treadmill training at MLSSw over a period of eight weeks. Muscle fiber-typing was determined by myosin ATPase histochemistry, citrate synthase activity by spectrophotometric assay, Ca(2+) handling-related protein levels by Western blot and reduced to oxidized glutathione ratio (GSH:GSSG) by high-performance liquid chromatography. Trained mice displayed higher running performance and citrate synthase activity compared with untrained mice. Improved running performance in trained mice was paralleled by fast-to-slow fiber-type shift and increased capillary density in both plantaris and soleus. Exercise training increased dihydropyridine receptor (DHPR) alpha 2 subunit, ryanodine receptor and Na(+)/Ca(2+) exchanger levels in plantaris and soleus. Moreover, exercise training elevated DHPR beta 1 subunit and sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) 1 levels in plantaris and SERCA2 levels in soleus of trained mice. Skeletal muscle GSH content and GSH:GSSG ratio was increased in plantaris and soleus of trained mice. Taken together, our findings indicate that MLSSw exercise-induced better running performance is, in part, due to increased levels of proteins involved in skeletal muscle Ca(2+) handling, whereas this response is partially dependent on specificity of skeletal muscle fiber-type composition. Finally, we demonstrated an augmented cellular redox status and GSH antioxidant capacity in trained mice.

Role of cholinergic, opioidergic and GABAergic neurotransmission of the dorsal hippocampus in the modulation of nociception in guinea pigs

Aims: Several physiological, pharmacological and behavioral lines of evidence suggest that the hippocampal formation is involved in nociception. The hippocampus is also believed to play an important role in the affective and motivational components of pain perception. Thus, Our aim was to investigate the participation of cholinergic, opioidergic and GABAergic systems of the dorsal hippocampus (DH) in the modulation of nociception in guinea pigs. Main methods: The test used consisted of the application of a peripheral noxious stimulus (electric shock) that provokes the emission of a vocalization response by the animal. Key findings: Our results showed that, in guinea pigs, microinjection of carbachol, morphine and bicuculline into the DH Promoted anti nociception, while muscimol promoted pronociception. These results were verified by a decrease and all increase, respectively, in the vocalization index in the vocalization test. This antinociceptive effect of carbachol (2.7 nmol) was blocked by previous administration of atropine (0.7 nmol) or naloxone (1.3 nmol) into the same site. In addition, the decrease in the vocalization index induced by the microinjection of morphine (2.2 nmol) into the DH was prevented by pretreatment with naloxone (1.3 nmol) or muscimol (0.5 nmol). At doses of 1.0 nmol, muscimol microinjection caused pronociception, while bicuculline promoted antinociception. Significance: These results indicate the involvement of the cholinergic, opioidergic and GABAergic systems of the DH in the modulation of antinociception in guinea pigs. In addition, the present study suggests that cholinergic transmission may activate the release of endorphins/enkephalin from interneurons of the DH, Which Would inhibit GABAergic neurons, resulting in antinociception. (C) 2008 Elsevier Inc. All rights reserved.

Histological and functional renal alterations caused by Bothrops alternatus snake venom: Expression and activity of Na(+)/K(+)-ATPase

Background: Acute renal failure is a serious complication of human envenoming by Bothrops snakes. The ion pump Na(+)/K(+)-ATPase has an important role in renal tubule function, where it modulates sodium reabsorption and homeostasis of the extracellular compartment. Here, we investigated the morphological and functional renal alterations and changes in Na(+)/K(+)-ATPase expression and activity in rats injected with Bothrops alternatus snake venom. Methods: Male Wistar rats were injected with venom (0.8 mg/kg, iv.) and renal function was assessed 6.24, 48 and 72 h and 7 days post-venom. The rats were then killed and renal Na(+)/K(+)-ATPase activity was assayed based on phosphate release from ATP; gene and protein expressions were assessed by real time PCR and immunofluorescence microscopy, respectively. Results: Venom caused lobulation of the capillary tufts, dilation of Bowman`s capsular space. F-actin disruption in Bowman`s capsule and renal tubule brush border, and deposition of collagen around glomeruli and proximal tubules that persisted seven days after envenoming. Enhanced sodium and potassium excretion, reduced proximal sodium reabsorption, and proteinuria were observed 6 h post-venom, followed by a transient decrease in the glomerular filtration rate. Gene and protein expressions of the Na(+)/K(+)-ATPase alpha(1) subunit were increased 6 h post-venom, whereas Na(+)/K(+)-ATPase activity increased 6 h and 24 h post-venom. Conclusions: Bothrops alternatus venom caused marked morphological and functional renal alterations with enhanced Na(+)/K(+)-ATPase expression and activity in the early phase of renal damage. General significance: Enhanced Na(+)/K(+)-ATPase activity in the early hours after envenoming may attenuate the renal dysfunction associated with venom-induced damage. (C) 2011 Elsevier B.V. All rights reserved.

Alpha7 Nicotinic Acetylcholine Receptor Expression and Activity During Neuronal Differentiation of PC12 Pheochromocytoma Cells

Nicotinic acetylcholine receptors (nAChR) exert pivotal roles in synaptic transmission, neuroprotection and differentiation. Particularly, homomeric alpha 7 receptors participate in neurite outgrowth, presynaptic control of neurotransmitter release and Ca(2+) influx. However, the study of recombinant alpha 7 nAChRs in transfected cell lines is difficult due to low expression of functional receptor channels. We show that PC12 pheochromocytoma cells induced to differentiation into neurons are an adequate model for studying differential nAChR gene expression and receptor activity. Whole-cell current recording indicated that receptor responses increased during the course of differentiation. Transcription of mRNAs coding for alpha 3, alpha 5, alpha 7, beta 2 and beta 4 subunits was present during the course of differentiation, while mRNAs coding for alpha 2, alpha 4 and beta 3 subunits were not expressed in PC12 cells. alpha 7 subunit expression was highest following 1 day of induction to differentiation. Activity of alpha 7 nAChRs, however, was most elevated on day 2 as revealed by inhibition experiments in the presence of 10 nM methyllycaconitine, rapid current decay and receptor responsiveness to the alpha 7 agonist choline. Increased alpha 7 receptor activity was noted when PC12 were induced to differentiation in the presence of choline, confirming that chronic agonist treatment augments nAChR activity. In summary, PC12 cells are an adequate model to study the role and pharmacological properties of this receptor during neuronal differentiation.

An alpha-type phospholipase A(2) inhibitor from Bothrops jararacussu snake plasma: Structural and functional characterization

An inhibitory protein that neutralizes the enzymatic, toxic and pharmacological activities of several phospholipases A(2) from Bothrops venoms was isolated from B. jararacussu snake plasma by affinity chromatography using the immobilized myotoxin BthTX-I on Sepharose gel. Biochemical characterization of this inhibitory protein, denominated alpha BjussuMIP, showed it to be an oligomeric glycoprotein with M-r of 24,000 for the monomeric subunit. Secondary structural analysis by circular dichroism revealed 44% alpha-helix, 18% beta-sheet, 10% beta-turn and 28% random coil structures. Circular dichroism spectroscopy indicated that no significant alterations in the secondary structure of either alpha BjussuMIP or the target protein occur following their interaction. The product from the reaction with reverse transcriptase produced a cDNA fragment of 432 bp that codifies for a mature protein of 144 amino acid residues. The first 21 amino acid residues from the N-terminal and five tryptic peptides were characterized by mass spectrometry of the mature protein and confirmed by the nucleotide sequence. Alignment of alpha BjussuMIP with other snake inhibitors showed a sequence similarity of 73-92% with these alpha PLIs. alpha BjussuMIP was relatively stable within the pH range of 6-12 and temperatures from 0 degrees C to 80 degrees C, even after deglycosylation. The results showed effects against Bothrops phospholipase A(2) activities (enzymatic, edema inducing, myotoxic, cytotoxic and bactericidal), suggesting that alpha BjussuMIP may prove useful in the treatment of snakebite envenomations. (C) 2008 Elsevier Masson SAS. All rights reserved.

Sequence variation of the alpha-lactalbumin gene in Holstein and Nellore cows

The alpha-lactalbumin is a subunit of lactose-synthase, an enzyme responsible for lactose production, a disaccharide that influences milk production. Sequence variations of bovine alpha-lactalbumin have been associated with differences in milk yield. This study aimed to analyze allelic frequency differences at position-1689 (g. AG) and+15 (g. AG) of the alpha-lactalbumin gene in Holstein (Bos taurus) and Nellore (Bos indicus) cows. Blood samples were analyzed from 34 Holstein, 104 Nellore, and 99 Dairy Nellore cows using PCR-RFLP. The different RFLP patterns were sequenced and a novel sequence variation on nucleotide-46 was identified. An adenine at this position was designated as the A allele and a guanine was designated B allele. The frequencies of alleles A-1689, A-46, and A+15 differed between Holstein and both Nellore breeds. The results show that differences in alpha-lactalbumin allelic variants in the 5`-flanking and the 5`-UTR region might be associated with differences in milk production between Holstein cows and cows from Nellore breeds. However, the lack of difference between Nellore and Dairy Nellore suggests that other sequence variantions that regulate milk production might be responsible for the selection of Dairy Nellore cows with superior milk production.

Light modulates the melanophore response to alpha-MSH in Xenopus laevis: An analysis of the signal transduction crosstalk mechanisms involved

Melanin granule (melanosome) dispersion within Xenopus laevis melanophores is evoked either by light or alpha-MSH. We have previously demonstrated that the initial biochemical steps of light and alpha-MSH signaling are distinct, since the increase in cAMP observed in response to alpha-MSH was not seen after light exposure. cAMP concentrations in response to alpha-MSH were significantly lower in cells pre-exposed to light as compared to the levels in dark-adapted melanophores. Here we demonstrate the presence of an adenylyl cyclase (AC) in the Xenopus melanophore, similar to the mammalian type IX which is inhibited by Ca(2+)-calmodulin-activated phosphatase. This finding supports the hypothesis that the cyclase could be negatively modulated by a light-promoted Ca(2+) increase. In fact, the activity of calcineurin PP2B phosphatase was increased by light, which could result in AC IX inhibition, thus decreasing the response to alpha-MSH. St-Ht31, a disrupting agent of protein kinase A (PKA)-anchoring kinase A protein (AKAP) complex totally blocked the melanosome dispersing response to alpha-MSH, but did not impair the photo-response in Xenopus melanophores. Sequence comparison of a melanophore AKAP partial clone with GenBank sequences showed that the anchoring protein was a gravin-like adaptor previously sequenced from Xenopus non-pigmentary tissues. Co-immunoprecipitation of Xenopus AKAP and the catalytic subunit of PKA demonstrated that PKA is associated with AKAP and it is released in the presence of alpha-MSH. We conclude that in X laevis melanophores, AKAP12 (gravin-like) contains a site for binding the inactive PKA thus compartmentalizing PKA signaling and also possesses binding sites for PKC. Light diminishes alpha-MSH-induced increase of cAMP by increasing calcineurin (PP2B) activity, which in turn inhibits adenylyl cyclase type IX, and/or by activating PKC, which phosphorylates the gravin-like molecule, thus destabilizing its binding to the cell membrane. (C) 2009 Elsevier Inc. All rights reserved.

Ric-8B interacts with G alpha olf and G gamma 13 and co-localizes with G alpha olf, G beta 1 and G gamma 13 in the cilia of olfactory sensory neurons

Olfactory sensory neurons are able to detect odorants with high sensitivity and specificity. We have demonstrated that Ric-8B, a guanine nucleotide exchange factor (GEF), interacts with G alpha olf and enhances odorant receptor signaling. Here we show that Ric-8B also interacts with G gamma 13, a divergent member of the G gamma subunit family which has been implicated in taste signal transduction, and is abundantly expressed in the cilia of olfactory sensory neurons. We show that G beta 1 is the predominant GP subunit expressed in the olfactory sensory neurons. Ric-8B and G beta 1, like G alpha olf and G gamma 13, are enriched in the cilia of olfactory sensory neurons. We also show that Ric-8B interacts with G alpha olf in a nucleotide dependent manner, consistent with the role as a GEF. Our results constitute the first example of a GEF protein that interacts with two different olfactory G protein subunits and further implicate Ric-8B as a regulator of odorant signal transduction. (C) 2008 Elsevier Inc. All rights reserved.

Antibody responses elicited in mice immunized with Bacillus subtilis vaccine strains expressing Stx2B subunit of enterohaemorragic Escherichia coli O157:H7

No effective vaccine or immunotherapy is presently available for patients with the hemolytic uremic syndrome (HUS) induced by Shiga-like toxin (Stx) producedbyenterohaemorragic Escherichia coli (EHEC) strains, such as those belonging to the O157:H7 serotype. In this work we evaluated the performance of Bacillus subtilis strains, a harmless spore former gram-positive bacterium species, as a vaccine vehicle for the expression of Stx2B subunit (Stx2B). A recombinant B. subtilis vaccine strain expressing Stx2B under the control of a stress inducible promoter was delivered to BALB/c mice via oral, nasal or subcutaneous routes using both vegetative cells and spores. Mice immunized with vegetative cells by the oral route developed low but specific anti-Stx2B serum IgG and fecal IgA responses while mice immunized with recombinant spores developed anti-Stx2B responses only after administration via the parenteral route. Nonetheless, serum anti-Stx2B antibodies raised in mice immunized with the recombinant B. subtilis strain did not inhibit the toxic effects of the native toxin, both under in vitro and in vivo conditions, suggesting that either the quantity or the quality of the induced immune response did not support an effective neutralization of Stx2 produced by EHEC strains.

Influence of alpha-tocopherol on the levels of serum anti-oxLDL antibodies

Purpose - This paper aims to evaluate the association between the a-tocopherol with the levels of serum anti-oxLDL autoantibodies and the risk markers for cardiovascular disease. Design/methodology/approach - A normolipidemic control group (n=30) and a hypercholesterolemic group (n=33) were used. Plasma lipid profile (colorimetric method), anti-oxLDL autoantibodies (ELISA) and a-tocopherol (HPLC) were analysed. Findings - The a-tocopherol (ß=-0.714; p=0.001) is negatively associated with anti-oxLDL autoantibodies in serum and with other risk markers for cardiovascular disease (BMI, WC, total cholesterol, LDL-c) and positively associated with HDL-c. Originality/value - Oxidized low density lipoprotein (oxLDL) and their autoantibodies are increased in subjects with hypercholesterolemia. The a-tocopherol can influence the levels of serum anti-oxLDL autoantibodies

Changes in Glucose and Glutamine Lymphocyte Metabolisms Induced by Type I Interferon alpha

In lymphocytes (LY), the well-documented antiproliferative effects of IFN-alpha are associated with inhibition of protein synthesis, decreased amino acid incorporation, and cell cycle arrest. However, the effects of this cytokine on the metabolism of glucose and glutamine in these cells have not been well investigated. Thus, mesenteric and spleen LY of male Wistar rats were cultured in the presence or absence of IFN-alpha, and the changes on glucose and glutamine metabolisms were investigated. The reduced proliferation of mesenteric LY was accompanied by a reduction in glucose total consumption (35%), aerobic glucose metabolism (55%), maximal activity of glucose-6-phosphate dehydrogenase (49%), citrate synthase activity (34%), total glutamine consumption (30%), aerobic glutamine consumption (20.3%) and glutaminase activity (56%). In LY isolated from spleen, IFN alpha also reduced the proliferation and impaired metabolism. These data demonstrate that in LY, the antiproliferative effects of IFN alpha are associated with a reduction in glucose and glutamine metabolisms.

INFLUENCE OF ALTITUDE, AGE AND DIAMETER ON YIELD AND ALPHA-BISABOLOL CONTENT OF CANDEIA TREES (Eremanthus erythropappus)

The heartwood of candeia tree is a source of essential oil rich in alpha-bisabolol, a substance widely used in the cosmetic and pharmaceutical industry. Bearing in mind the economic importance of alpha-bisabolol, this work aimed to evaluate the influence of tree age on the yield and content of alpha-bisabolol present in essential oil from candeia, considering two distinct reliefs and three diameter classes, in Aiuruoca region, south Minas Gerais state. The two distinct reliefs correspond respectively to one section of the stand growing at 1,000m of altitude (Area 1) and another section growing at 1,100m of altitude (Area 2). In each section, 15 trees were felled from among 3 different diameter classes. Discs were removed from the base of each tree to estimate their age by doing growth ring count. Soil samples were taken and Subjected to physical and chemical analysis. The logs were reduced into chips and random samples were taken for distillation to extract essential oil. The method used was steam distillation at a pressure of 2 kgf/cm(2)/2.5 h. The chemical analysis was performed in a gas chromatograph (GC) based on the alpha-bisabolol standard reference. The yield of essential oil from trees in Area I was higher than that from trees in Area 2, with the same pattern of influence for older trees. In Area 2, the alpha-bisabolol content was higher in younger trees. No differences were found between the relevant parameters in relation to diameter classes.

Heat and chemical stress modulate the expression of the alpha-RYR gene in broiler chickens

The biological cause of Pork Stress syndrome, which leads to PSE (pale, soft, exudative) meat, is excessive release of Ca(2+) ions, which is promoted by a genetic mutation in the ryanodine receptors (RyR) located in the sarcoplasmic reticulum of the skeletal muscle cells. We examined the relationship between the formation of PSE meat under halothane treatment and heat stress exposure in chicken alpha RYR hot spot fragments. Four test groups were compared: 1) birds slaughtered without any treatment, i.e., the control group (C); 2) birds slaughtered immediately after halothane treatment (H); 3) birds slaughtered immediately after heat stress treatment (HS), and 4) birds exposed to halothane and to heat stress (H+HS), before slaughtering. Breast muscle mRNA was extracted, amplified by RT-PCR, and sequenced. PSE meat was evaluated using color determination (L*value). The most common alteration was deletion of a single nucleotide, which generated a premature stop codon, resulting in the production of truncated proteins. The highest incidence of nonsense transcripts came with exposure to halothane; 80% of these abnormal transcripts were detected in H and H+HS groups. As a consequence, the incidence of abnormal meat was highest in the H+HS group (66%). In HS, H, and C groups, PSE meat developed in 60, 50, and 33% of the samples, respectively. Thus, halothane apparently modulates alpha RYR gene expression in this region, and synergically with exposure to heat stress, causes Avian Stress syndrome, resulting in PSE meat in broiler chickens.

Immunomodulatory effects of recombinant BCG expressing pertussis toxin on TNF-alpha and IL-10 in a bladder cancer model

Background: Since successful treatment of superficial bladder cancer with BCG requires proper induction of Th1 immunity, we have developed a rBCG-S1PT strain that induced a stronger cellular immune response than BCG. This preclinical study was designed to compare the modulatory effects of BCG and rBCG-S1PT on bladder TNF-alpha and IL-10 expression and to evaluate antitumour activity. Methods: For Experiment I, the MB49 bladder cancer cell line was used in C57BL/6 mice. Chemical cauterization of the bladder was performed to promote intravesical tumor implantation. Mice were treated by intravesical instillation with BCG, rBCG-S1PT or PBS once a week for four weeks. After 35 days the bladders were removed and weighed. TNF-