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Highlights • Valerenic acid modulates the neuropathological markers of Parkinson´s disease. • Valerenic acid attenuates neuroinflammation in a Parkinson´s disease model. • Astrocytes and cytokines are targets in the anti-inflammatory effect of valerenic acid. • The molecular docking confirms the interaction of valerenic acid with 5-HT5A receptors., Parkinson´s disease is the most important neuromotor pathology due to the prominent loss of dopaminergic neurons in the substantia nigra pars compacta. There is an inherent deficiency of dopamine in Parkinson´s disease, which is aggravated when neuroinflammatory processes are present. Several biomolecules are interesting candidates for the regulation of inflammation and possible neuroprotection, such as valerenic acid, one of the main components of Valeriana officinalis. A 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP)-induced mouse model of Parkinson's disease was developed to evaluate the motor effects of valerenic acid. The evaluation was carried out with four tests (an invert screen test for muscle strength, cross beam test, open field mobility test and lifting on hind legs test). Subsequently, the neuroinflammatory process was evaluated through ELISA of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α and IFN-γ). The decreases in the inflammatory and neurodegenerative processes were evaluated by Western blot and immunohistochemistry analyses of the tissues, which included an evaluation of the tyrosine hydroxylase and GFAP proteins. Finally, the predicted mechanism of action of valerenic acid was supported by molecular docking calculations with the 5-HT5A receptor. The results indicate that the use of valerenic acid as a co-treatment decreases the neuroinflammation in Parkinson's disease induced by MPTP and provides evidence of a decrease in the evaluated pro-inflammatory cytokines and in the amount of GFAP in the mesencephalic area. Valerenic acid prevents neuroinflammation in a Parkinson's disease mouse model, which might reflect the neuroprotection of dopaminergic neurons with the recovery of motor ability.

Multidrug resistance protein-4 (MRP4) belongs to the ABC transporter superfamily and promotes the transport of xenobiotics including drugs. A non-synonymous single nucleotide polymorphisms (nsSNPs) in the ABCC4 gene can promote changes in the structure and function of MRP4. In this work, the interaction of certain endogen substrates, drug substrates, and inhibitors with wild type-MRP4 (WT-MRP4) and its variants G187W and Y556C were studied to determine differences in the intermolecular interactions and affinity related to SNPs using protein threading modeling, molecular docking, all-atom, coarse grained, and umbrella sampling molecular dynamics simulations (AA-MDS and CG-MDS, respectively). The results showed that the three MRP4 structures had significantly different conformations at given sites, leading to differences in the docking scores (DS) and binding sites of three different groups of molecules. Folic acid (FA) had the highest variation in DS on G187W concerning WT-MRP4. WT-MRP4, G187W, Y556C, and FA had different conformations through 25 ns AA-MD. Umbrella sampling simulations indicated that the Y556C-FA complex was the most stable one with or without ATP. In Y556C, the cyclic adenosine monophosphate (cAMP) and ceefourin-1 binding sites are located out of the entrance of the inner cavity, which suggests that both cAMP and ceefourin-1 may not be transported. The binding site for cAMP and ceefourin-1 is quite similar and the affinity (binding energy) of ceefourin-1 to WT-MRP4, G187W, and Y556C is greater than the affinity of cAMP, which may suggest that ceefourin-1 works as a competitive inhibitor. In conclusion, the nsSNPs G187W and Y556C lead to changes in protein conformation, which modifies the ligand binding site, DS, and binding energy.

The neuroendocrine transdifferentiation has been found in many cancer cell types, such as prostate, lung and gastrointestinal cells and is accompanied by a lower patient life expectancy. The transdifferentiation process has been induced in vitro by the exposure to different stimuli in human lung adenocarcinoma. The aim of this work was to identify the morphological characteristics of the neuroendocrine phenotype in a human lung cancer cell line, induced by two cAMP elevating agents (IBMX and FSK). Our results showed two phenotypes, one produced by IBMX with higher volume, cell size and increased number of secondary projections, and the other produced by FSK with higher area, roughness of the membrane, cell neurite percentage, number of outgrowths per cell and increased number of primary projections. In conclusion, we describe some morphological and ultrastructural characteristics of the neuroendocrine phenotype in A549 human lung cancer cell line promoted by IBMX and FSK to contribute to the understanding of the autocrine or paracrine signaling within the tumor microenvironment.

The excess of many chemical substances in humans, such as copper has been reported to induce DNA damage and trigger cancer development...

Background and Objectives: Prostate cancer is the second most harmful disease in men worldwide and the number of cases is increasing. Therefore, new natural agents with anticancer potential should be examined and the response of existing therapeutic drugs must be enhanced. Stevia pilosa and Stevia eupatoria are two species that have been widely used in traditional medicine, but their effectiveness on cancer cells and their interaction with antineoplastic drugs have not been studied. The aim of this study was to evaluate the anticancer activity of Stevia pilosa methanolic root extract (SPME) and Stevia eupatoria methanolic root extract (SEME) and their effect, combined with enzalutamide, on prostate cancer cells. Materials and Methods: The study was conducted on a human fibroblast cell line, and on androgen-dependent (LNCaP) and androgen-independent (PC-3) prostate cancer cell lines. The cell viability was evaluated using a Trypan Blue exclusion test for 48 h, and the migration by a wound-healing assay for 24, 48, and 72 h. Results: The results indicate that SPME and SEME were not cytotoxic at concentrations less than 1000 &mu, g/mL in the human fibroblasts. SPME and SEME significantly reduced the viability and migration of prostate cancer cells in all concentrations evaluated. The antiproliferative effect of the Stevia extracts was higher in cancer cells than in normal cells. The enzalutamide decreased the cell viability in all concentrations tested (10&ndash, 50 &micro, M). The combination of the Stevia extracts and enzalutamide produced a greater effect on the inhibition of the proliferation and migration of cancer cells than the Stevia extracts alone, but not of the enzalutamide alone. Conclusion: The results indicate that SPME and SEME have an inhibitory effect on the viability and migration of prostate cancer cells and do not interfere with the enzalutamide anticancer effect. The data suggest that Stevia extracts may be a potential source of molecules for cancer treatment.

It is known that growth hormone (GH) is expressed in immune cells, where it exerts immunomodulatory effects. However, the mechanisms of expression and release of GH in the immune system remain unclear. We analyzed the effect of growth hormone-releasing hormone (GHRH), thyrotropin-releasing hormone (TRH), ghrelin (GHRL), and somatostatin (SST) upon GH mRNA expression, intracellular and released GH, Ser133-phosphorylation of CREB (pCREBS133), intracellular Ca2+ levels, as well as B-cell activating factor (BAFF) mRNA expression in bursal B-lymphocytes (BBLs) cell cultures since several GH secretagogues, as well as their corresponding receptors (-R), are expressed in B-lymphocytes of several species. The expression of TRH/TRH-R, ghrelin/GHS-R1a, and SST/SST-Rs (Subtypes 1 to 5) was observed in BBLs by RT-PCR and immunocytochemistry (ICC), whereas GHRH/GHRH-R were absent in these cells. We found that TRH treatment significantly increased local GH mRNA expression and CREB phosphorylation. Conversely, SST decreased GH mRNA expression. Additionally, when added together, SST prevented TRH-induced GH mRNA expression, but no changes were observed in pCREBS133 levels. Furthermore, TRH stimulated GH release to the culture media, while SST increased the intracellular content of this hormone. Interestingly, SST inhibited TRH-induced GH release in a dose-dependent manner. The coaddition of TRH and SST decreased the intracellular content of GH. After 10 min. of incubation with either TRH or SST, the intracellular calcium levels significantly decreased, but they were increased at 60 min. However, the combined treatment with both peptides maintained the Ca2+ levels reduced up to 60-min. of incubation. On the other hand, BAFF cytokine mRNA expression was significantly increased by TRH administration. Altogether, our results suggest that TRH and SST are implicated in the regulation of GH expression and release in BBL cultures, which also involve changes in pCREBS133 and intracellular Ca2+ concentration. It is likely that TRH, SST, and GH exert autocrine/paracrine immunomodulatory actions and participate in the maturation of chicken BBLs.

Dementia caused by Alzheimer's disease (AD) is mainly characterized by accumulation in the brain of extra- and intraneuronal amyloid-β (Aβ) and tau proteins, respectively, which selectively affect specific regions, particularly the neocortex and the hippocampus. Sporadic AD is mainly caused by an increase in apolipoprotein E, a component of chylomicrons, which are cholesterol transporters in the brain. Recent studies have shown that high lipid levels, especially cholesterol, are linked to AD. Adenosine is an atypical neurotransmitter that regulates a wide range of physiological functions by activating four P1 receptors (A1, A2A, A2B, and A3) and P2 purinergic receptors that are G protein-coupled. A1 receptors are involved in the inhibition of neurotransmitter release, which could be related to AD. The aim of the present work was to study the effects of a lard-enriched diet (LED) on cognitive and memory processes in adult rats (6 months of age) as well as the effect of theobromine on these processes. The results indicated that the fat-enriched diet resulted in a long-term deterioration in cognitive and memory functions. Increased levels of Aβ protein and IL-1β were also observed in the rats fed with a high-cholesterol diet, which were used to validate the AD animal model. In addition, the results of qPCR and immunohistochemistry indicated a decrease in gene expression and distribution of A1 purinegic receptor, respectively, in the hippocampus of LED-fed rats. Interestingly, theobromine, at both concentrations tested, restored A1 receptor levels and improved cognitive functions and Aβ levels for a dose of 30 mg/L drinking water.

T cell acute lymphoblastic leukaemia is a type of cancer that develops from lymphoid progenitors, and chemotherapy is corner stone of the treatment. Thiopurine drugs, consisting of 6-mercaptopurine, 6-thioguanine, and azathioprine (Aza), can effectively treat this disease. To be activated, Aza must first be biotransformed to 6-mercaptopurine by thiol groups in glutathione. However, glutathione is decreased in cancer cells due to high levels of reactive oxygen species (ROS). N-acetylcysteine could provide thiol groups for glutathione synthesis to biotransform Aza. Using flow cytometry, the ability of N-acetylcysteine to increase the antiproliferative and apoptotic effects of Aza without increasing ROS was tested in Jurkat cells. Individually, Aza 1.0 and 2.0 μM, as well as N- acetylcysteine 3.0 mM, induced apoptosis and cell cycle arrest. Together, Aza + N-acetylcysteine significantly reduced proliferation compared to that obtained with the individual drugs. Combination of N-acetylcysteine 3.0 mM with Aza 1.0 μM was as effective at inducing apoptosis as Aza 2.0 μM alone. The combination of N-acetylcysteine 3.0 mM + Aza 1.0 μM increased cell cycle arrest at the G2/M phase. We found that Aza 1.0 or 2.0 μM induced a significant increase in ROS compared to that in untreated cells, while N-acetylcysteine 3.0 mM and N-acetylcysteine 3.0 mM + Aza 1.0 μM kept ROS at control levels; the latter drugpairing represents a favourable combination to reduce oxidative stress in the presence of Aza. In conclusion, N-acetylcysteine augments antiproliferative and apoptotic effects of Aza without increasing ROS in vitro.

The sodium-coupled neutral amino acid transporter 2 (SNAT2) is a protein that is expressed ubiquitously in mammalian tissues and that displays Na(+), voltage and pH dependent activity. This transporter mediates the passage of small zwitterionic amino acids across the cell membrane and regulates the cell homeostasis and its volume. We have examined the expression of SNAT2 mRNA and protein during the development of the rat cerebral cortex, from gestation through the postnatal stages to adulthood. Our data reveal that SNAT2 mRNA and protein expression is higher during embryogenesis, while it subsequently diminishes during postnatal development. Moreover, during embryonic period SNAT2 colocalizes with the radial glial cells marker GLAST, while in postnatal period it is mainly detected in neuronal dendrites. These findings suggest a relevant role for amino acid transport through SNAT2 in the developing embryonic brain.

Common beans ( Phaseolus vulgaris L.) contain a high proportion of undigested carbohydrates (NDC) that can be fermented in the large intestine to produce short-chain fatty acids (SCFA) such as acetate, propionate, and butyrate. The objective of the present study was to evaluate the composition and chemopreventive effect of a polysaccharide extract (PE) from cooked common beans ( P. vulgaris L) cv. Negro 8025 on azoxymethane (AOM) induced colon cancer in rats. The PE induced SCFA production with the highest butyrate concentrated in the cecum zone: 6.7 +/- 0.06 mmol/g of sample for PE treatment and 5.29 +/- 0.24 mmol/g of sample for PE + AOM treatment. The number of aberrant crypt foci (ACF) and the transcriptional expression of bax and caspase-3 were increased, and rb expression was decreased. The data suggest that PE decreased ACF and had an influence on the expression of genes involved in colon cancer for the action of butyrate concentration.

Aerial parts of Medicago sativa L. have been used as food and its consumption has been associated with health benefits, one among the most important being menopausal symptoms control. This work was aimed to explore possible pharmacological effects of two new alfalfa-derived products that have recently emerged as daily beverage preparations. In exploring their potential estrogenic effects, they produced no relevant alteration in the uterus. However, lowering glucose levels until normal values without causing further hypoglycemic effect were observed, when rats were treated with 1.5 g/kg/day samples. In vivo acute toxicity was not found when the alfalfa products were tested up to 3 g/kg rat weight. Furthermore, in vitro studies were conducted to assess their possible toxic effects. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase tests were carried out on the Caco-2 cell model to determine cell viability and membrane integrity. A concentration-dependent effect was observed, with a significant decrease in cell viability after exposure to concentrations of alfalfa product up to 100 mg/mL (after 3 h of incubation) and 50 mg/mL (after 24 h of treatment). Although in vitro level, the decrease in cell viability at these still low doses may underlie some toxicity, making necessary additional studies before any recommendation of a sustained consumption of these products by humans. info:eu-repo/semantics/publishedVersion

Glycosylation is one of the post-translational modifications that growth hormone (GH) can undergo. This has been reported for human, rat, mouse, pig, chicken and buffalo GH. The nature and significance of GH glycosylation remains to be elucidated. This present study further characterizes glycosylated chicken GH (G-cGH) and examines changes in the pituitary concentration of G-cGH during embryonic development and post hatching growth. G-cGH was purified from chicken pituitaries by affinity chromatography (Concanavalin A-Sepharose and monoclonal antibody bound to Sepharose). Immunoreactive G-cGH has a MW of 26 kDa or 29 kDa as determined by SDS-PAGE, respectively, under non-reducing and reducing conditions. Evidence that it is N-glycosylated comes from its susceptibility to peptide N-glycosidase F, and its resistance to O-glycosidase. Based on the ability of G-cGH to bind Concanavalin A or wheat germ agglutinin but not other lectins and its susceptibility to peptide N-glycosidase F, a hybrid or biantennary type glycopeptide (GlcNac2, Man) structure is proposed. Some G-cGH can be observed in the pituitary at most ages examined (from 15-day embryo to adult). Moreover, electron microscopy revealed the presence of both immuno-reactive GH and Concanavalin A-reactive sites in the same secretory granules in the somatotrope. There were marked changes in the level and relative proportion of G-cGH in the pituitary gland during development and growth, the proportion of G-cGH rising during late embryonic development (e.g., between 15 and 18 days of development) and with further increases between 9 weeks and 15 weeks old. G-cGH was able to bind to chicken liver membrane preparations with less affinity than non-glycosylated monomer; on the other hand, however, G-cGH stimulated cell proliferation on Nb2 lymphoma bioassay whereas the non-glycosylated monomer was uncapable to do it.

Serotonin is an ancient molecular signal and a recognized neurotransmitter brainwide distributed with particular presence in hippocampus. Almost all serotonin receptor subtypes are expressed in hippocampus, which implicates an intricate modulating system, considering that they can be localized as autosynaptic, presynaptic, and postsynaptic receptors, even colocalized within the same cell and being target of homo- and heterodimerization. Neurons and glia, including immune cells, integrate a functional network that uses several serotonin receptors to regulate their roles in this particular part of the limbic system.

Hydroxytryptamine (5-HT) is involved in a variety of different physiological processes and behaviors through the activation of equally diverse receptors subtypes. In this work we studied the changes on the expression of 5-HT5A receptors in rat hippocampus induced by leptin, an adipocyte-derived hor- mone that has been reported to participate in the modulation of food intake and in adult hippocampal neurogenesis. To study the effect of leptin on the 5-HT5A receptor gene expression a qRT-PCR was used and the distribution of those receptors in the hippocampus was visualized by immunohistochemistry. Rats were separated in four groups: control (untreated rats), leptin-treated, serotonin-treated and lep- tin + serotonin treated. The results showed that even though the 5-HT5A gene expression did not change in the hippocampus of any of the treated groups, in the rats treated with leptin and serotonin, the specific immunostaining for the 5-HT5A serotonin receptor decreased significantly in the dentate gyrus. © 2010 Elsevier Ireland Ltd. All rights reserved.

The distribution of the glycine receptor subunits alpha1-3 and beta in the developing rat cerebellum was studied from postnatal day 1 to adulthood by means of quantitative RT-PCR and immunohistochemistry. qRT-PCR of postnatal cerebella indicated the presence of mRNA for each subunit, with a relative expression of alpha2alpha3alpha1beta. The immunohistochemistry indicated a strong alpha2 signal in the Purkinje cells, internal and external granular layers. The alpha1-3 subunits had weak signals in the Purkinje cells and molecular layer. The alpha1 subunit was expressed at a low level and was also found in the white matter. The function of these receptors in neuronal and glial plasma membranes in early postnatal development remains to be determined.

There is evidence for a cleaved form of GH in the chicken pituitary gland. A 25 kDa band of immunoreactive-(ir-)GH, as well as the 22 kDa monomeric form and some oligomeric forms were observed when purified GH or fresh pituitary extract were subjected to SDS-PAGE under nonreducing conditions. Under reducing conditions, the 25 kDa ir-GH was no longer observed, being replaced by a 15 kDa band, consistent with reduction of the disulfide bridges of the cleaved form. The type of protease involved was investigated using exogenous proteases and monomeric cGH. Cleaved forms of chicken GH were generated by thrombin or collagenase. The site of cleavage was found in position Arg133-Gly134 as revealed by sequencing the fragments produced. The NH2-terminal sequence of 40 amino acid residues in the 15 kDa form was identical to that of the rcGH and analysis of the remaining 7 kDa fragment showed an exact identity with positions 134-140 of cGH structure. The thrombin cleaved GH and the 15 kDa form showed reduced activity (0.8% and 0.5% of GH, respectively) in a radioreceptor assay employing a chicken liver membrane preparation. However, this fragment had a clear bioactivity in an angiogenic bioassay and was capable to inhibit the activity of deiodinase type III in the chicken liver.