Extracellular ATP triggers proteolysis and cytosolic Ca2+ rise in Plasmodium berghei and Plasmodium yoelii malaria parasites

Background: Plasmodium has a complex cell biology and it is essential to dissect the cell-signalling pathways underlying its survival within the host. Methods: Using the fluorescence resonance energy transfer (FRET) peptide substrate Abz-AIKFFARQ-EDDnp and Fluo4/AM, the effects of extracellular ATP on triggering proteolysis and Ca2+ signalling in Plasmodium berghei and Plasmodium yoelii malaria parasites were investigated. Results: The protease activity was blocked in the presence of the purinergic receptor blockers suramin (50 mu M) and PPADS (50 mu M) or the extracellular and intracellular calcium chelators EGTA (5 mM) and BAPTA/AM (25, 100, 200 and 500 mu M), respectively for P. yoelii and P. berghei. Addition of ATP (50, 70, 200 and 250 mu M) to isolated parasites previously loaded with Fluo4/AM in a Ca2+-containing medium led to an increase in cytosolic calcium. This rise was blocked by pre-incubating the parasites with either purinergic antagonists PPADS (50 mu M), TNP-ATP (50 mu M) or the purinergic blockers KN-62 (10 mu M) and Ip5I (10 mu M). Incubating P. berghei infected cells with KN-62 (200 mu M) resulted in a changed profile of merozoite surface protein 1 (MSP1) processing as revealed by western blot assays. Moreover incubating P. berghei for 17 h with KN-62 (10 mu M) led to an increase in rings forms (82% +/- 4, n = 11) and a decrease in trophozoite forms (18% +/- 4, n = 11). Conclusions: The data clearly show that purinergic signalling modulates P. berghei protease(s) activity and that MSP1 is one target in this pathway.

Characterization of yolk sac proteins of Bos indicus cattle embryos

The yolk sac is an embryonic membrane that is essential for the embryo's initial survival in many mammals. It also plays an important role in the production of proteins necessary for development. We studied proteins of the yolk sac in bovine embryos at up to 40 days of gestation. We examined the yolk sac of 17 bovine embryos at different gestational periods, measuring a-fetoprotein, alpha-1-antitrypsin, and transferrin. This experiment was carried out by Western blot technique, associated with electrophoresis on a 6% sodium dodecyl sulfate polyacrylamide gel. Mouse monoclonal antibody anti-human-alpha-fetoprotein, mouse antibody anti-human-transferrin and rabbit polyclonal anti-human-alpha-1-antitrypsin were used as primary antibodies, and conjugated peroxidase as a secondary antibody. We detected the three proteins in some of the yolk sac samples; however, the bands in some specimens (samples) were weak, maybe a result of poor antigen-antibody reaction, since the antibodies used in this study were not specific to bovine proteins. The fact that weak bands appeared might be due to a weak cross-reaction.

Hemolymph ion regulation and kinetic characteristics of the gill (Na+, K+)-ATPase in the hermit crab Clibanarius vittatus (Decapoda, Anomura) acclimated to high salinity

We examine hemolymph ion regulation and the kinetic properties of a gill microsomal (Na+, K+)-ATPase from the intertidal hermit crab, Clibanarius vittatus, acclimated to 45 parts per thousand salinity for 10 days. Hemolymph osmolality is hypo-regulated (1102.5 +/- 22.1 mOsm kg(-1) H2O) at 45 parts per thousand but elevated compared to fresh-caught crabs (801.0 +/- 40.1 mOsm kg(-1) H2O). Hemolymph [Na+ (323.0 +/- 2.5 mmol L-1) and [Me2+) (34.6 +/- 1.0 mmol L-1) are hypo-regulated while [Ca2+] (22.5 +/- 0.7 mmol L-1) is hyper-regulated; [K+] is hyper-regulated in fresh-caught crabs (17.4 +/- 0.5 mmol L-1) but hypo-regulated (6.2 +/- 0.7 mmol L-1) at 45 parts per thousand. Protein expression patterns are altered in the 45 parts per thousand-acclimated crabs, although Western blot analyses reveal just a single immunoreactive band, suggesting a single (Na+, K+)-ATPase alpha-subunit isoform, distributed in different density membrane fractions. A high-affinity (Vm = 46.5 +/- 3.5 U mg(-1); K-0.5 = 7.07 +/- 0.01 mu mol L-1) and a low-affinity ATP binding site (Vm = 108.1 +/- 2.5 U mg(-1); K-0.5 = 0.11 +/- 0.3 mmol L-1), both obeying cooperative kinetics, were disclosed. Modulation of (Na+, K+)-ATPase activity by Mg2+, K+ and NH4+ also exhibits site-site interactions, but modulation by Na+ shows Michaelis-Menten kinetics. (Na+, K+)-ATPase activity is synergistically stimulated up to 45% by NH4+ plus K+. Enzyme catalytic efficiency for variable [K+] and fixed [NH4+] is 10-fold greater than for variable [NH4+] and fixed [K+]. Ouabain inhibited approximate to 80% of total ATPase activity (K-I=464.7 +/- 23.2 mu mol L-1), suggesting that ATPases other than (Na+, K+)-ATPase are present. While (Na+, K+)-ATPase activities are similar in fresh-caught (around 142 nmol Pi min(-1) mg(-1)) and 45 parts per thousand-acclimated crabs (around 154 nmol Pi min(-1) mg(-1)), ATP affinity decreases 110-fold and Na+ and K+ affinities increase 2-3-fold in 45 parts per thousand-acclimated crabs. (C) 2012 Elsevier Inc. All rights reserved.

Human immunodeficiency virus prevalence, incidence, and residual risk of transmission by transfusions at Retrovirus Epidemiology Donor Study-II blood centers in Brazil

BACKGROUND: In Brazil nationally representative donor data are limited on human immunodeficiency virus (HIV) prevalence, incidence, and residual transfusion risk. The objective of this study was to analyze HIV data obtained over 24 months by the Retrovirus Epidemiology Donor Study-II program in Brazil. STUDY DESIGN AND METHODS: Donations reactive to third-and fourth-generation immunoassays (IAs) were further confirmed by a less-sensitive (LS) IA algorithm and Western blot (WB). Incidence was calculated for first-time (FT) donors using the LS-EIA results and for repeat donors with a model developed to include all donors with a previous negative donation. Residual risk was projected by multiplying composite FT and repeat donor incidence rates by HIV marker-negative infectious window periods. RESULTS: HIV prevalence among FT donors was 92.2/ 105 donations. FT and repeat donor and composite incidences were 38.5 (95% confidence interval [CI], 25.651.4), 22.5 (95% CI, 17.6-28.0), and 27.5 (95% CI, 22.0-33.0) per 100,000 person-years, respectively. Male and community donors had higher prevalence and incidence rates than female and replacement donors. The estimated residual risk of HIV transfusion transmission was 11.3 per 106 donations (95% CI, 8.4-14.2), which could be reduced to 4.2 per 106 donations (95% CI, 3.2-5.2) by use of individual-donation nucleic acid testing (NAT). CONCLUSION: The incidence and residual transfusion risk of HIV infection are relatively high in Brazil. Implementation of NAT will not be sufficient to decrease transmission rates to levels seen in the United States or Europe; therefore, other measures focused on decreasing donations by at-risk individuals are also necessary.

Co-Stimulation of PAFR and CD36 Is Required for oxLDL-Induced Human Macrophages Activation

The oxidative process of LDL particles generates molecules which are structurally similar to platelet-activating factor (PAF), and some effects of oxidized LDL (oxLDL) have been shown to be dependent on PAF receptor (PAFR) activation. In a previous study, we showed that PAFR is required for upregulation of CD36 and oxLDL uptake. In the present study we analyzed the molecular mechanisms activated by oxLDL in human macrophages and the contribution of PAFR to this response. Human adherent monocytes/macrophages were stimulated with oxLDL. Uptake of oxLDL and CD36 expression were determined by flow cytometry; MAP kinases and Akt phosphorylation by Western blot; IL-8 and MCP-1 concentration by ELISA and mRNA expression by real-time PCR. To investigate the participation of the PI3K/Akt pathway, G alpha i-coupled protein or PAFR, macrophages were treated with LY294002, pertussis toxin or with the PAFR antagonists WEB2170 and CV3988, respectively before addition of oxLDL. It was found that the addition of oxLDL to human monocytes/macrophages activates the PI3K/Akt pathway which in turn activates the MAPK (p38 and JNK). Phosphorylation of Akt requires the engagement of PAFR and a G alpha i-coupled protein. The upregulation of CD36 protein and the uptake of oxLDL as well as the IL-8 production are dependent on PI3K/Akt pathway activation. The increased CD36 protein expression is dependent on PAFR and G alpha i-coupled protein. Transfection studies using HEK 293t cells showed that oxLDL uptake occurs with either PAFR or CD36, but IL-8 production requires the co-transfection of both PAFR and CD36. These findings show that PAFR has a pivotal role in macrophages response to oxLDL and suggest that pharmacological intervention at the level of PAFR activation might be beneficial in atherosclerosis.

Differential expression of HINT1 in schizophrenia brain tissue

Recent findings in the literature suggest a relation between histidine triad nucleotide-binding protein-1 (HINT1) and psychiatric disorders such as major depression, anxiety, and schizophrenia, although its physiological roles are not completely comprehended. Using Western blot, we compared HINT1 protein expression in the postmortem dorsolateral prefrontal cortex and thalamus of schizophrenia patients and healthy controls for contributing to elucidate the role of HINT1 in schizophrenia pathophysiology. HINT1 was found to be downregulated in the dorsolateral prefrontal cortex and upregulated in the thalamus. Our results combined to previous studies in human samples and preclinical models support the notion that HINT1 must be more explored as a potential target for psychiatric disorders.

Glucocorticoid and Estrogen Receptors Are Reduced in Mitochondria of Lung Epithelial Cells in Asthma

Mitochondrial glucocorticoid (mtGR) and estrogen (mtER) receptors participate in the coordination of the cell's energy requirement and in the mitochondrial oxidative phosphorylation enzyme (OXPHOS) biosynthesis, affecting reactive oxygen species (ROS) generation and induction of apoptosis. Although activation of mtGR and mtER is known to trigger anti-inflammatory signals, little information exists on the presence of these receptors in lung tissue and their role in respiratory physiology and disease. Using a mouse model of allergic airway inflammation disease and applying confocal microscopy, subcellular fractionation, and Western blot analysis we showed mitochondrial localization of GR alpha and ER beta in lung tissue. Allergic airway inflammation caused reduction in mtGR alpha, mtER beta, and OXPHOS enzyme biosynthesis in lung cells mitochondria and particularly in bronchial epithelial cells mitochondria, which was accompanied by decrease in lung mitochondrial mass and induction of apoptosis. Confirmation and validation of the reduction of the mitochondrial receptors in lung epithelial cells in human asthma was achieved by analyzing autopsies from fatal asthma cases. The presence of the mitochondrial GR alpha and ER beta in lung tissue cells and especially their reduction in bronchial epithelial cells during allergic airway inflammation suggests a crucial role of these receptors in the regulation of mitochondrial function in asthma, implicating their involvement in the pathophysiology of the disease.

Generation and Biological Properties of a Recombinant Dodecahedron Containing the Short Fiber Protein of the Human Adenovirus 41

Objective: In order to gain further insight into the function of the enteric adenovirus short fiber (SF), we have constructed a recombinant dodecahedron containing the SF protein of HAdV-41 and the HAdV-3 penton base. Methods: Recombinant baculoviruses expressing the HAdV-41 SF protein and HAdV-3 penton base were cloned and amplified in Sf9 insect cells. Recombinant dodecahedra were expressed by coinfection of High Five (TM) cells with both baculoviruses, 72 h post-infection. Cell lysate was centrifuged on sucrose density gradient and the purified recombinant dodecahedra were recovered. Results: Analysis by negative staining electron microscopy demonstrated that chimeric dodecahedra made of the HAdV-3 penton base and decorated with the HAdV-41 SF were successfully generated. Next, recombinant dodecahedra were digested with pepsin and analyzed by Western blot. A 'site-specific' proteolysis of the HAdV-41 SF was observed, while the HAdV-3 penton base core was completely digested. Conclusion: These results show that, in vitro, the HAdV-41 SF likely undergoes proteolysis in the gastrointestinal tract, its natural environment, which may facilitate the recognition of receptors in intestinal cells. The results obtained in the present study may be the basis for the development of gene therapy vectors towards the intestinal epithelium, as well as orally administered vaccine vectors, but also for the HAdV-41 SF partner identification. Copyright (C) 2011 S. Karger AG, Basel

Efficient RNAi-induced Protein Knockdown in Somatic Cells Using Diced or Chemically Produced Small Interfering RNAs (siRNA)

Background: RNA interference (RNAi) is a post-transcriptional gene silencing process in which double-stranded RNA (dsRNA) directs the degradation of a specific corresponding target mRNA. The mediators of this process are small dsRNAs of approximately 21 to 23 bp in length, called small interfering RNAs (siRNAs), which can be prepared in vitro and used to direct the degradation of specific mRNAs inside cells. Hence, siRNAs represent a powerful tool to study and control gene and cell function. Rapid progress has been made in the use of siRNA as a means to attenuate the expression of any protein for which the cDNA sequence is known. Individual siRNAs can be chemically synthesized, in vitro-transcribed, or expressed in cells from siRNA expression vectors. However, screening for the most efficient siRNAs for post-transcriptional gene silencing in cells in culture is a laborious and expensive process. In this study, the effectiveness of two siRNA production strategies for the attenuation of abundant proteins for DNA repair were compared in human cells: (a) the in vitro production of siRNA mixtures by the Dicer enzyme (Diced siRNAs); and (b) the chemical synthesis of very specific and unique siRNA sequences (Stealth RNai (TM)). Materials, Methods & Results: For in vitro-produced siRNAs, two segments of the human Ku70 (167 bp in exon 5; and 249 bp in exon 13; NM001469) and Xrcc4 (172 bp in exon 2; and 108 bp in exon 6; NM003401) genes were chosen to generate dsRNA for subsequent "Dicing" to create mixtures of siRNAs. The Diced fragments of siRNA for each gene sequence were pooled and stored at -80 degrees C. Alternatively, chemically synthesized Stealth siRNAs were designed and generated to match two very specific gene sequence regions for each target gene of interest (Ku70 and Xrcc4). HCT116 cells were plated at 30% confluence in 24- or 6-well culture plates. The next day, cells were transfected by lipofection with either Diced or Stealth siRNAs for Ku70 or Xrcc4, in duplicate, at various doses, with blank and sham transfections used as controls. Cells were harvested at 0, 24, 48, 72 and 96 h post-transfection for protein determination. The knockdown of specific targeted gene products was quantified by Western blot using GAPDH as control. Transfection of gene-specific siRNA to either Ku70 or Xrcc4 with both Diced and Stealth siRNAs resulted in a down regulation of the targeted proteins to approximately 10 to 20% of control levels 48 h after transfection, with recovery to pre-treatment levels by 96 h. Discussion: By transfecting cells with Diced or chemically synthesized Stealth siRNAs, Ku70 and Xrcc4, two highly expressed proteins in cells, were effectively attenuated, demonstrating the great potential for the use of both siRNA production strategies as tools to perform loss of function experiments in mammalian cells. In fact, down-regulation of Ku70 and Xrcc4 has been shown to reduce the activity of the non-homologous end joining DNA pathway, a very desirable approach for the use of homologous recombination technology for gene targeting or knockout studies. Stealth RNAi (TM) was developed to achieve high specificity and greater stability when compared with mixtures of enzymatically-produced (Diced) siRNA fragments. In this study, both siRNA approaches inhibited the expression of Ku70 and Xrcc4 gene products, with no detectable toxic effects to the cells in culture. However, similar knockdown effects using Diced siRNAs were only attained at concentrations 10-fold higher than with Stealth siRNAs. The application of RNAi technology will expand and continue to provide new insights into gene regulation and as potential applications for new therapies, transgenic animal production and basic research.

Neural mobilization reverses behavioral and cellular changes that characterize neuropathic pain in rats

Background: The neural mobilization technique is a noninvasive method that has proved clinically effective in reducing pain sensitivity and consequently in improving quality of life after neuropathic pain. The present study examined the effects of neural mobilization (NM) on pain sensitivity induced by chronic constriction injury (CCI) in rats. The CCI was performed on adult male rats, submitted thereafter to 10 sessions of NM, each other day, starting 14 days after the CCI injury. Over the treatment period, animals were evaluated for nociception using behavioral tests, such as tests for allodynia and thermal and mechanical hyperalgesia. At the end of the sessions, the dorsal root ganglion (DRG) and spinal cord were analyzed using immunohistochemistry and Western blot assays for neural growth factor (NGF) and glial fibrillary acidic protein (GFAP). Results: The NM treatment induced an early reduction (from the second session) of the hyperalgesia and allodynia in CCI-injured rats, which persisted until the end of the treatment. On the other hand, only after the 4th session we observed a blockade of thermal sensitivity. Regarding cellular changes, we observed a decrease of GFAP and NGF expression after NM in the ipsilateral DRG (68% and 111%, respectively) and the decrease of only GFAP expression after NM in the lumbar spinal cord (L3-L6) (108%). Conclusions: These data provide evidence that NM treatment reverses pain symptoms in CCI-injured rats and suggest the involvement of glial cells and NGF in such an effect.

Reduced platelet amyloid precursor protein ratio (APP ratio) predicts conversion from mild cognitive impairment to Alzheimer's disease

Studies have shown that platelet APP ratio (representing the percentage of 120-130 kDa to 110 kDa isoforms of the amyloid precursor protein) is reduced in patients with mild cognitive impairment (MCI) and Alzheimer's disease (AD). In the present study, we sought to determine if baseline APP ratio predicts the conversion from MCI to AD dementia after 4 years of longitudinal assessment. Fifty-five older adults with varying degrees of cognitive impairment (34 with MCI and 21 with AD) were assessed at baseline and after 4 years. MCI patients were re-classified according to the conversion status upon follow-up: 25 individuals retained the diagnostic status of MCI and were considered as stable cases (MCI-MCI); conversely, in nine cases the diagnosis of dementia due to AD was ascertained. The APP ratio (APPr) was determined by the Western blot method in samples of platelets collected at baseline. We found a significant reduction of APPr in MCI patients who converted to dementia upon follow-up. These individuals had baseline APPr values similar to those of demented AD patients. The overall accuracy of APPr to identify subjects with MCI who will progress to AD was 0.74 +/- A 0.10, p = 0.05. The cut-off of 1.12 yielded a sensitivity of 75 % and a specificity of 75 %. Platelet APPr may be a surrogate marker of the disease process in AD, with potential implications for the assessment of abnormalities in the APP metabolism in patients with and at risk for dementia. However, diagnostic accuracy was relatively low. Therefore, studies in larger samples are needed to determine whether APPr may warrant its use as a biomarker to support the early diagnosis of AD.

An in vitro study showing the three-dimensional microenvironment influence over the behavior of head and neck squamous cell carcinoma

Objectives: The Head and Neck Squamous Cell Carcinoma (HNSCC) ranks sixth worldwide. The mechanisms of growth, invasion and metastasis of this pathology are extensively studied and generally related to specific variations in signaling pathways like the PI3K-Akt; however most of these competent studies have been performed bidimensionally, which may hide important questions. This study sought to analyze the influence of the microenvironment upon the behavior of HNSCC. Study Design: The status of pAkt, NF-kappa B and Cyclin D1 proteins was accessed through immunofluorescence and western blot methods in HNSCC cell lines originating from tongue, pharynx and metastatic lymph node when submitted to a three-dimensional culture model utilizing a matrix system. A bidimensional culture model (monolayer) was used as control. Results: The HNSCC cell lines cultured three-dimensionally exhibited a growth pattern characterized by small isolated islands, different from the control group. When the three-dimensional model was applied, two of the studied cell lines showed the same expression pattern as the bidimensional model regarding nuclear or cytoplasmatic localization, as well as reduction of all protein levels; however, the cell line originated from tongue, which specially has the epidermal growth factor receptor constitutively activated, demonstrated nuclear translocation of pAkt and also an increase in the levels of Cyclin D1. Conclusions: The results suggest the influence of the microenvironment upon the behavior of HNSCC cells due to the changed expression of proteins related to tumor growth and cellular invasion. Furthermore, intrinsically genetic conditions also played important roles over the cells, despite the culture model employed.

Isolated total RNA and protein are preserved after thawing for more than twenty-four hours

OBJECTIVE: The preservation of biological samples at a low temperature is important for later biochemical and/or histological analyses. However, the molecular viability of thawed samples has not been studied sufficiently in depth. The present study was undertaken to evaluate the viability of intact tissues, tissue homogenates, and isolated total RNA after defrosting for more than twenty-four hours. METHODS: The molecular viability of the thawed samples (n = 82) was assessed using the A260/A280 ratio, the RNA concentration, the RNA integrity, the level of intact mRNA determined by reverse transcriptase polymerase chain reaction, the protein level determined by Western blotting, and an examination of the histological structure. RESULTS: The integrity of the total RNA was not preserved in the thawed intact tissue, but the RNA integrity and level of mRNA were perfectly preserved in isolated defrosted samples of total RNA. Additionally, the level of beta-actin protein was preserved in both thawed intact tissue and homogenates. CONCLUSION: Isolated total RNA does not undergo degradation due to thawing for at least 24 hours, and it is recommended to isolate the total RNA as soon as possible after tissue collection. Moreover, the protein level is preserved in defrosted tissues.

Neutrophil dysfunction induced by hyperglycemia: modulation of myeloperoxidase activity

Our data suggest that impaired activity of myeloperoxidase (MPO) may play an important role in the dysfunction of neutrophils from hyperglycemic rats. Neutrophil biochemical pathways include the NADPH oxidase system and the MPO enzyme. They both play important role in the killing function of neutrophils. The effect of hyperglycemia on the activity of these enzymes and the consequences with regard to Candida albicans phagocytosis and the microbicidal property of rat peritoneal neutrophils is evaluated here. The NADPH oxidase system activity was measured using chemiluminescence and cytochrome C reduction assays. MPO activity was measured by monitoring HOCl production, and MPO protein expression was analysed using Western blot and immunofluorescence. C. albicans phagocytosis and death were evaluated by optical microscopy using the MayGrunwaldGiemsa staining method. ROS generation kinetic was slightly delayed in the diabetic group. MPO expression levels were higher in diabetic neutrophils; however, MPO activity was decreased in these same neutrophils compared with the controls. C. albicans phagocytosis and killing were lower in the diabetic neutrophils. Based on our experimental model, the phagocytic and killing functions of neutrophil phagocytosis are impaired in diabetic rats because of the decreased production of HOCl, highlighting the importance of MPO in the microbicidal function of neutrophils. Copyright (c) 2012 John Wiley & Sons, Ltd.

Stimulation of peripheral Kappa opioid receptors inhibits inflammatory hyperalgesia via activation of the PI3K gamma/AKT/nNOS/NO signaling pathway

Background: In addition to their central effects, opioids cause peripheral analgesia. There is evidence showing that peripheral activation of kappa opioid receptors (KORs) inhibits inflammatory pain. Moreover, peripheral mu-opioid receptor (MOR) activation are able to direct block PGE(2)-induced ongoing hyperalgesia However, this effect was not tested for KOR selective activation. In the present study, the effect of the peripheral activation of KORs on PGE(2)-induced ongoing hyperalgesia was investigated. The mechanisms involved were also evaluated. Results: Local (paw) administration of U50488 (a selective KOR agonist) directly blocked, PGE(2)-induced mechanical hyperalgesia in both rats and mice. This effect was reversed by treating animals with L-NMMA or N-propyl-L-arginine (a selective inhibitor of neuronal nitric oxide synthase, nNOS), suggesting involvement of the nNOS/NO pathway. U50488 peripheral effect was also dependent on stimulation of PI3K gamma/AKT because inhibitors of these kinases also reduced peripheral antinociception induced by U50488. Furthermore, U50488 lost its peripheral analgesic effect in PI3K gamma null mice. Observations made in vivo were confirmed after incubation of dorsal root ganglion cultured neurons with U50488 produced an increase in the activation of AKT as evaluated by western blot analyses of its phosphorylated form. Finally, immunofluorescence of DRG neurons revealed that KOR-expressing neurons also express PI3K gamma (congruent to 43%). Conclusions: The present study indicates that activation of peripheral KORs directly blocks inflammatory hyperalgesia through stimulation of the nNOS/NO signaling pathway which is probably stimulated by PI3K gamma/AKT signaling. This study extends a previously study of our group suggesting that PI3K gamma/AKT/nNOS/NO is an important analgesic pathway in primary nociceptive neurons.