Metformin and soybean-derived bioactive molecules attenuate the expansion of stem cell-like epithelial subpopulation and confer apoptotic sensitivity in human colon cancer cells

Colorectal cancer (CRC) is a disease whose genesis may include metabolic dysregulation. Cancer stem cells are attractive targets for therapeutic interventions since their aberrant expansion may underlie tumor initiation, progression, and recurrence. To investigate the actions of metabolic regulators on cancer stem cell-like cells (CSC) in CRC, we determined the effects of soybean-derived bioactive molecules and the anti-diabetes drug metformin (MET), alone and together, on the growth, survival, and frequency of CSC in human HCT116 cells. Effects of MET (60 μM) and soybean components genistein (Gen, 2 μM), lunasin (Lun, 2 μM), β-conglycinin (β-con, 3 μM), and glycinin (Gly, 3 μM) on HCT116 cell proliferation, apoptosis, and mRNA/protein expression and on the frequency of the CSC CD133(+)CD44(+) subpopulation by colonosphere assay and fluorescence-activated cell sorting/flow cytometry were evaluated. MET, Gen, and Lun, individually and together, inhibited HCT116 viability and colonosphere formation and, conversely, enhanced HCT116 apoptosis. Reductions in frequency of the CSC CD133(+)CD44(+) subpopulation with MET, Gen, and Lun were found to be associated with increased PTEN and reduced FASN expression. In cells under a hyperinsulinemic state mimicking metabolic dysregulation and without and with added PTEN-specific inhibitor SF1670, colonosphere formation and frequency of the CD133(+)CD44(+) subpopulation were decreased by MET, Lun and Gen, alone and when combined. Moreover, MET + Lun + Gen co-treatment increased the pro-apoptotic and CD133(+)CD44(+)-inhibitory efficacy of 5-fluorouracil under hyperinsulinemic conditions. Results identify molecular networks shared by MET and bioavailable soy food components, which potentially may be harnessed to increase drug efficacy in diabetic and non-diabetic patients with CRC.

Effects of nitric oxide synthesis inhibitor or fluoxetine treatment on depression-like state and cardiovascular changes induced by chronic variable stress in rats

Comorbidity between mood disorders and cardiovascular disease has been described extensively. However, available antidepressants can have cardiovascular side effects. Treatment with selective inhibitors of neuronal nitric oxide synthase (nNOS) induces antidepressant effects, but whether the antidepressant-like effects of these drugs are followed by cardiovascular changes has not been previously investigated. Here, we tested in male rats exposed to chronic variable stress (CVS) the hypothesis that nNOS blockers are advantageous compared with conventional antidepressants in terms of cardiovascular side effects. We compared the effects of chronic treatment with the preferential nNOS inhibitor 7-nitroindazole (7-NI) with those evoked by the conventional antidepressant fluoxetine on alterations that are considered as markers of depression (immobility in the forced swimming test, FST, decreased body weight gain and increased plasma corticosterone concentration) and cardiovascular changes caused by CVS. Rats were exposed to a 14-day CVS protocol, while being concurrently treated daily with either 7-NI (30 mg/kg) or fluoxetine (10 mg/kg). Fluoxetine and 7-NI prevented the increase in immobility in the FST induced by CVS and reduced plasma corticosterone concentration in stressed rats. Both these treatments also prevented the CVS-evoked reduction of the depressor response to vasodilator agents and baroreflex changes. Fluoxetine and 7-NI-induced cardiovascular changes independent of stress exposure, including cardiac autonomic imbalance, increased intrinsic heart rate and vascular sympathetic modulation, a reduction of the pressor response to vasoconstrictor agents, and impairment of baroreflex activity. Altogether, these findings provide evidence that fluoxetine and 7-NI have similar effects on the depression-like state induced by CVS and on cardiovascular function.

Toll-Like recepctors (TLRs) and retinoic acid inducible gene – I (RIG-I) activation by viral analogs in bovine endometrial cells

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Glucose,cholesterol,total proteins and insulin-like growth factor typeI values in the follicular fluid of female river buffaloes (Bubalus bubalis)

Twenty six Murrah female river buffaloes, between 45 and 70 d post-partum, empty, multiparae, with an average live weight of 675 ± 56 kg, and average body condition of 3.5 points, in a 1 to 5 scale, were used to determine the concentrations of glucose, cholesterol, total protein and insulin-like growth factor type I(IGF-I) in the follicular fluid. The fluid was collected from dominant follicles, with diameters between 8 and 12 mm, by in vivo follicular aspiration. The oestrous cycle stage was not taken into account. The wave of follicular development was synchronized six days prior to the collection. Biochemical analyses of glucose and cholesterol were performed by the enzymatic colorimetric method with the utilization of commercial kits of Glicose (GOD-PAP) and Cholesterol (CHOD-PAP) (Kovalent), respectively. For the determination of total protein, the commercial kit total Protein (Kovalent), method Biuret, was employed. Readings were carried out through absorption spectrophotometry with visible light. Through the radioimmunoanalysis (RIA) technique the concentration of IGF-I was obtained using commercial kits of IRMA IGF-I (IMMUNOTECH). Descriptive statistics was used, by applying the PROC MEANS procedure of the SAS (2009) statistical package. Glucose concentrations (4.0 ± 0.75 mmol/L) and IGF-I (340 ± 129.83 ng/mL) showed higher values in female river buffaloes and dairy cows regarding those reported in other studies. However, cholesterol levels (0.51 ± 0.12 mmol/L) and total proteins (58.4 ± 4.43 g/L) were lower. Results indicate that there is a relationship between the concentration of biochemical indicators, the nutritional aspects, the diameter of the aspired follicles and the productive period.

Estudo da resistência à corrosão de ligas Ni-Cr-Mo-Ta E Ni-Cr-Mo-W em solução NaF 0,08 mol/L, pH 4,7 para aplicações odontológicas

A large number of metal alloys are used in Dentistry for the manufacture of fixed and removable dentures. In the oral cavity, these structures are exposed to a chemically aggressive medium, like saliva and mechanical efforts, like mastication. In addition, acidic solutions containing fluoride ions are also frequently used in dental treatments to prevent dental plates and decays development. In this context, it was considered important to investigate the influence that a fourth element could exert when added to the ternary alloy Ni-Cr-Mo, largely used in Brazil. Therefore, electrochemical tests were done to evaluate the resistance to corrosion of quaternary alloy 65Ni-25Cr-5Mo-5Ta and 65Ni-25Cr-5Mo-5W in NaF solution 0,08mol / L, pH = 4.7. For greater understanding the microstructure and morphology of alloys were studied, through metallographic analysis, using optics microscopy and electron microscopy scanning. For the electrochemical tests were applied techniques traditionally used in corrosion researches, such as: potential measures in open circuit (OCP) and cyclic polarization (CP). It was found that both quaternary alloys showed very similar results. Comparing these quaternary alloys with the ternary 65Ni-25Cr-10Mo, it was found that the quaternary alloys exhibit greater resistance to corrosion, in other words, less passivation current density than the ternary alloy, showing that it is advantageous to add a fourth element in the alloy

Expressão gênica e protéica de receptores Toll-like em células do sangue periférico materno de gestações normais e complicadas por prematuridade

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Estudo da resistência à corrosão de ligas Ni-Cr-Mo-Ta E Ni-Cr-Mo-W em solução NaF 0,08 mol/L, pH 4,7 para aplicações odontológicas

A large number of metal alloys are used in Dentistry for the manufacture of fixed and removable dentures. In the oral cavity, these structures are exposed to a chemically aggressive medium, like saliva and mechanical efforts, like mastication. In addition, acidic solutions containing fluoride ions are also frequently used in dental treatments to prevent dental plates and decays development. In this context, it was considered important to investigate the influence that a fourth element could exert when added to the ternary alloy Ni-Cr-Mo, largely used in Brazil. Therefore, electrochemical tests were done to evaluate the resistance to corrosion of quaternary alloy 65Ni-25Cr-5Mo-5Ta and 65Ni-25Cr-5Mo-5W in NaF solution 0,08mol / L, pH = 4.7. For greater understanding the microstructure and morphology of alloys were studied, through metallographic analysis, using optics microscopy and electron microscopy scanning. For the electrochemical tests were applied techniques traditionally used in corrosion researches, such as: potential measures in open circuit (OCP) and cyclic polarization (CP). It was found that both quaternary alloys showed very similar results. Comparing these quaternary alloys with the ternary 65Ni-25Cr-10Mo, it was found that the quaternary alloys exhibit greater resistance to corrosion, in other words, less passivation current density than the ternary alloy, showing that it is advantageous to add a fourth element in the alloy

Expressão gênica e protéica de receptores Toll-like em células do sangue periférico materno de gestações normais e complicadas por prematuridade

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Acetic acid- and phenyl-p-benzoquinone-induced overt pain-like behavior depends on spinal activation of MAP kinases, PI3K and microglia in mice

The acetic acid and phenyl-p-benzoquinone are easy and fast screening models to access the activity of novel candidates as analgesic drugs and their mechanisms. These models induce a characteristic and quantifiable overt pain-like behavior described as writhing response or abdominal contortions. The knowledge of the mechanisms involved in the chosen model is a crucial step forward demonstrating the mechanisms that the candidate drug would inhibit because the mechanisms triggered in that model will be addressed. Herein, it was investigated the role of spinal mitogen-activated protein (MAP) kinases ERK (extracellular signal-regulated kinase), JNK (Jun N-terminal Kinase) and p38, PI3K (phosphatidylinositol 3-kinase) and microglia in the writhing response induced by acetic acid and phenyl-p-benzoquinone, and flinch induced by formalin in mice. Acetic acid and phenyl-p-benzoquinone induced significant writhing response over 20 min. The nociceptive response in these models were significantly and in a dose-dependent manner reduced by intrathecal pre-treatment with ERK (PD98059), JNK (SB600125), p38 (SB202190) or PI3K (wortmannin) inhibitors. Furthermore, the co-treatment with MAP kinase and PI3K inhibitors, at doses that were ineffective as single treatment, significantly inhibited acetic acid- and phenyl-p-benzoquinone-induced nociception. The treatment with microglia inhibitors minocycline and fluorocitrate also diminished the nociceptive response. Similar results were obtained in the formalin test. Concluding. MAP kinases and PI3K are important spinal signaling kinases in acetic acid and phenyl-p-benzoquinone models of overt pain-like behavior and there is also activation of spinal microglia indicating that it is also important to determine whether drugs tested in these models also modulate such spinal mechanisms. (C) 2012 Elsevier Inc. All rights reserved.

Common chromosomal imbalances and stemness-related protein expression markers in endometriotic lesions from different anatomical sites: the potential role of stem cells

Endometriosis is a multifactorial gynecological disease characterized by the presence of functional endometrium-like tissue in ectopic sites. Several studies have focused on elucidating the immunological, endocrine, environmental and genetic factors involved in endometriosis. However, its pathogenesis is still unclear. High-resolution comparative genomic hybridization was applied to screen for genomic imbalances in laser microdissected stromal and epithelial cells from 20 endometriotic lesions and three samples of eutopic endometrium derived from eight patients. The expression of seven stemness-related markers (CD9, CD13, CD24, CD34, CD133, CD117/c-Kit and Oct-4) in endometrial tissue samples was evaluated by immunohistochemistry. Samples of eutopic endometrium showed normal genomic profiles. In ectopic tissues, an average of 68 genomic imbalances was detected per sample. DNA losses were more frequently detected and involved mainly 3p, 5q, 7p, 9p, 11q, 16q, 18q and 19q. Many of the genomic imbalances detected were common to endometriotic stroma and epithelia and also among different endometriotic sites from the same patient. These findings suggested a clonal origin of the endometriotic cells and the putative involvement of stem cells. Positive immunostaining for CD9, CD34, c-Kit and Oct-4 markers was detected in isolated epithelial and/or stromal cells in eutopic and ectopic endometrium in the majority of cases. The presence of shared genomic alterations in stromal and epithelial cells from different anatomical sites of the same patient and the expression of stemness-related markers suggested that endometriosis arises as a clonal proliferation with the putative involvement of stem cells.

Lyophilized bovine colostrum as a source of immunoglobulins and insulin-like growth factor for newborn goat kids

The objective of this study was to evaluate lyophilized bovine colostrum as an alternative source of passive immunity and insulin like growth factor I (IGF-I) for goat kids, considering newborns consuming non-maternal colostrum. Twenty-nine male newborns received 5% of body weight of lyophilized bovine (LBC) or goat colostrum (GC), both with 55 mg/mL of IgG, at 0, 7 and 14 h of life. Blood samples were collected at 0, 7, 14, 18, 24, 36, 48, 72 and 96 h of life to determine serum IgG, total protein (TP), IGF-I and apparent efficiency of IgG absorption at 7, 14, 18 and 24 h (AEA(7hr), AEA(14hr), AEA(18hr), AEA(total), respectively). In LBC, the values of serum IgG at 14, 18,24 and 48 h (13.1, 13.4, 14.1 and 14.6 mg/mL, respectively) were higher than the values at 0 and 7 h (0.04 and 6.9 mg/mL, respectively). In GC, the serum IgG at 18 h (9.3 mg/mL) was higher than the value at 7 h (5.5 mg/mL). AEA(7hr) and AEA(14hr) in LBC were the same (19.2 and 18.5%, respectively, P>0.05) and the values of AEA(18hr) and AEA(total), 9.3 and 9.5%, respectively, were equal and smaller than AEA(7hr), and AEA(14hr). In GC, AEA(7hr), 20.8%, was higher than AEA(14hr), 16.1% (P<0.05) and AEA(18hr), and AEA(total), 9.2 and 8.0%, respectively, were equal and smaller than AEA(7hr) and AEA(14hr). The serum TP and IGF-I were not affected by colostrum feeding. Considering the variables study in the present work, lyophilized bovine colostrum constitutes a promising alternative substitute to goat colostrum in newborn goat kids, since the supply of immunoglobulins and IGF-I was suitable for the kids. (c) 2012 Elsevier B.V. All rights reserved.

Differential expression of toll-like receptor mRNAs in recurrent aphthous ulceration

BACKGROUND: Toll-like receptors (TLR) are membrane proteins that recognize conserved molecules derived from bacterial, viral, fungal or host tissues. They are responsible for promoting the production of cytokines and chemokines, increasing the expression of costimulatory molecules and influencing the T Helper response (Th) toward either a Th1 or Th2 profile, thereby modulating the regulatory T cell response and controlling the integrity of the epithelial barrier. The key factors responsible for increased susceptibility to recurrent aphthous ulceration (RAU) are unclear, and because TLRs are involved in both immune regulation and control of the epithelial barrier, a deficiency in TLR activity is likely to cause increased susceptibility. METHODS: We investigated the gene expression of TLRs one through 10 in tissue samples and peripheral blood mononuclear cells (PBMC) of RAU patients in comparison to healthy controls using real-time quantitative reverse transcription PCR. RESULTS: The analysis of mRNA expression levels in oral lesion showed significant (P < 0.01) overexpression of the TLR2(similar to 6-fold) gene and decreased expression of the TLR3 (similar to 5-fold) and TLR5 (similar to 6-fold) genes in comparison with healthy oral mucosa. The analysis of mRNA expression in PBMC indicated a down-regulation of TLR5 gene expression in the cells from RAU patients (P < 0.05; similar to 2-fold). CONCLUSION: Our results support the hypothesis that a subset of RAU patients has fewer TLR expression that have been tentatively implicated in antiinflammatory effects. This derangement of TLR gene expression may cause an overlay exuberant inflammation reaction in situations where normal individuals are resistant. J Oral Pathol Med (2012) 41: 8085

Gastrin-Releasing Peptide Receptor Antagonism Induces Protection from Lethal Sepsis: Involvement of Toll-like Receptor 4 Signaling

In sepsis, toll-like receptor (TLR)-4 modulates the migration of neutrophils to infectious foci, favoring bacteremia and mortality. In experimental sepsis, organ dysfunction and cytokines released by activated macrophages can be reduced by gastrin-releasing peptide (GRP) receptor (GRPR) antagonist RC-3095. Here we report a link between GRPR and TLR-4 in experimental models and in sepsis patients. RAW 264.7 culture cells were exposed to lipopolysaccharide (LPS) or tumor necrosis factor (TNF)-alpha and RC-3095 (10 ng/mL), Male Wistar rats were subjected to cecal ligation and puncture (CLP), and RC-3095 was administered (3 mg/kg, subcutaneously); after 6 h, we removed the blood, bronchoalveolar lavage, peritoneal lavage and lung. Human patients with a clinical diagnosis of sepsis received a continuous infusion with RC-3095 (3 mg/kg, intravenous) over a period of 12 h, and plasma was collected before and after RC-3095 administration and, in a different set of patients with systemic inflammatory response syndrome (SIRS) or sepsis. GRP plasma levels were determined. RC-3095 inhibited TLR-4, extracellular-signal-related kinase (ERK)-1/2, Jun NH2-terminal kinase (JNK) and Akt and decreased activation of activator protein 1 (AP-1), nuclear factor (NF)-kappa B and interleukin (IL)-6 in macrophages stimulated by LPS. It also decreased IL-6 release from macrophages stimulated by TNF-alpha. RC-3095 treatment in CLP rats decreased lung TLR-4, reduced the migration of cells to the lung and reduced systemic cytokines and bacterial dissemination. Patients with sepsis and systemic inflammatory response syndrome have elevated plasma levels of GRP which associates with clinical outcome in the sepsis patients. These findings highlight the role of GRPR signaling in sepsis outcome and the beneficial action of GRPR antagonists in controlling the inflammatory response in sepsis through a mechanism involving at least inhibition of TLR-4 signaling. Online address: http://www.molmed.org doi: 10.2119/molmed.2012.00083

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

Structural insights regarding an insecticidal Talisia esculenta protein and its biotechnological potential for Diatraea saccharalis larval control

Talisin is a seed-storage protein from Talisia esculenta that presents lectin-like activities, as well as proteinase-inhibitor properties. The present study aims to provide new in vitro and in silico biochemical information about this protein, shedding some light on its mechanistic inhibitory strategies. A theoretical three-dimensional structure of Talisin bound to trypsin was constructed in order to determine the relative interaction mode. Since the structure of non-competitive inhibition has not been elucidated, Talisin-trypsin docking was carried out using Hex v5.1, since the structure of non-competitive inhibition has not been elucidated. The predicted non-coincidence of the trypsin binding site is completely different from that previously proposed for Kunitz-type inhibitors, which demonstrate a substitution of an Arg(64) for the Glu(64) residue. Data, therefore, provide more information regarding the mechanisms of non-competitive plant proteinase inhibitors. Bioassays with Talisin also presented a strong insecticide effect on the larval development of Diatraea saccharalis, demonstrating LD50 and ED50 of ca. 2.0% and 1.5%, respectively. (C) 2011 Elsevier Inc. All rights reserved.