Regulation of Multidrug Resistance Proteins by Genistein in a Hepatocarcinoma Cell Line: Impact on Sorafenib Cytotoxicity

Hepatocellular carcinoma (HCC) is the fifth most frequent cancer worldwide. Sorafenib is the only drug available that improves the overall survival of HCC patients. P-glycoprotein (P-gp), Multidrug resistance-associated proteins 2 and 3 (MRP2 and 3) and Breast cancer resistance protein (BCRP) are efflux pumps that play a key role in cancer chemoresistance. Their modulation by dietary compounds may affect the intracellular accumulation and therapeutic efficacy of drugs that are substrates of these transporters. Genistein (GNT) is a phytoestrogen abundant in soybean that exerts its genomic effects through Estrogen-Receptors and Pregnane-X-Receptor (PXR), which are involved in the regulation of the above-mentioned transporters. We evaluated the effect of GNT on the expression and activity of P-gp, MRP2, MRP3 and BCRP in HCC-derived HepG2 cells. GNT (at 1.0 and 10 μM) increased P-gp and MRP2 protein expression and activity, correlating well with an increased resistance to sorafenib cytotoxicity as detected by the methylthiazole tetrazolium (MTT) assay. GNT induced P-gp and MRP2 mRNA expression at 10 but not at 1.0 μM concentration suggesting a different pattern of regulation depending on the concentration. Induction of both transporters by 1.0 μM GNT was prevented by cycloheximide, suggesting translational regulation. Downregulation of expression of the miR-379 by GNT could be associated with translational regulation of MRP2. Silencing of PXR abolished P-gp induction by GNT (at 1.0 and 10 μM) and MRP2 induction by GNT (only at 10 μM), suggesting partial mediation of GNT effects by PXR. Taken together, the data suggest the possibility of nutrient-drug interactions leading to enhanced chemoresistance in HCC when GNT is ingested with soy rich diets or dietary supplements.     

EBNA2 Binds to Genomic Intervals Associated with Multiple Sclerosis and Overlaps with Vitamin D Receptor Occupancy

Epstein-Barr virus (EBV) is a non-heritable factor that associates with multiple sclerosis (MS). However its causal relationship with the disease is still unclear. The virus establishes a complex co-existence with the host that includes regulatory influences on gene expression. Hence, if EBV contributes to the pathogenesis of MS it may do so by interacting with disease predisposing genes. To verify this hypothesis we evaluated EBV nuclear antigen 2 (EBNA2, a protein that recent works by our and other groups have implicated in disease development) binding inside MS associated genomic intervals. We found that EBNA2 binding occurs within MS susceptibility sites more than expected by chance (factor of observed vs expected overlap [O/E] = 5.392-fold, p < 2.0e-05). This remains significant after controlling for multiple genomic confounders. We then asked whether this observation is significant per se or should also be viewed in the context of other disease relevant gene-environment interactions, such as those attributable to vitamin D. We therefore verified the overlap between EBNA2 genomic occupancy and vitamin D receptor (VDR) binding sites. EBNA2 shows a striking overlap with VDR binding sites (O/E = 96.16-fold, p < 2.0e-05), even after controlling for the chromatin accessibility state of shared regions (p <0.001). Furthermore, MS susceptibility regions are preferentially targeted by both EBNA2 and VDR than by EBNA2 alone (enrichment difference = 1.722-fold, p = 0.0267). Taken together, these findings demonstrate that EBV participates in the gene-environment interactions that predispose to MS.     

The Local Complement Activation on Vascular Bed of Patients with Systemic Sclerosis: A Hypothesis-Generating Study

ObjectiveThe role of complement system in the pathogenesis of systemic sclerosis (SSc) has been debated during the last decade but an evident implication in this disease has never been found. We carried out an explorative study on SSc patients to evaluate the expression of soluble and local C5b-9 complement complex and its relation with a complement regulator, the Membrane Cofactor Protein (MCP, CD46) on skin vascular bed as target distinctive of SSc disease. We also analyzed two polymorphic variants in the complement activation gene cluster involving the MCP region.MethodsC5b-9 plasma levels of SSc patients and healthy subjects were analyzed by ELISA assay. Archival skin biopsies of SSc patients and controls were subjected to immunofluorescence analysis to detect C5b-9 and MCP on vascular endothelial cells. The expression of MCP was validated by immunoblot analysis with specific antibody. Polymorphic variants in the MCP gene promoter were tested by a quantitative PCR technique-based allelic discrimination method.ResultsEven though circulating levels of C5b-9 did not differ between SSc and controls, C5b-9 deposition was detected in skin biopsies of SSc patients but not in healthy subjects. MCP was significantly lower in skin vessels of SSc patients than in healthy controls and was associated with the over-expression of two polymorphic variants in the MCP gene promoter, which has been related to more aggressive phenotypes in other immune-mediated diseases.ConclusionsOur results firsty document the local complement activation with an abnormal expression of MCP in skin vessels of SSc patients, suggesting that a subset of SSc patients might be exposed to more severe organ complications and clinical evolution due to abnormal local complement activation.     

Ligand Binding to the FA3-FA4 Cleft Inhibits the Esterase-Like Activity of Human Serum Albumin

The hydrolysis of 4-nitrophenyl esters of hexanoate (NphOHe) and decanoate (NphODe) by human serum albumin (HSA) at Tyr411, located at the FA3-FA4 site, has been investigated between pH 5.8 and 9.5, at 22.0°C. Values of K _s, k ₊₂, and k ₊₂/K _s obtained at [HSA] ≥ 5×[NphOXx] and [NphOXx] ≥ 5×[HSA] (Xx is NphOHe or NphODe) match very well each other; moreover, the deacylation step turns out to be the rate limiting step in catalysis (i.e., k ₊₃ << k ₊₂). The pH dependence of the kinetic parameters for the hydrolysis of NphOHe and NphODe can be described by the acidic pK _a-shift of a single amino acid residue, which varies from 8.9 in the free HSA to 7.6 and 7.0 in the HSA:NphOHe and HSA:NphODe complex, respectively; the pK>_a-shift appears to be correlated to the length of the fatty acid tail of the substrate. The inhibition of the HSA-Tyr411-catalyzed hydrolysis of NphOHe, NphODe, and 4-nitrophenyl myristate (NphOMy) by five inhibitors (i.e., diazepam, diflunisal, ibuprofen, 3-indoxyl-sulfate, and propofol) has been investigated at pH 7.5 and 22.0°C, resulting competitive. The affinity of diazepam, diflunisal, ibuprofen, 3-indoxyl-sulfate, and propofol for HSA reflects the selectivity of the FA3-FA4 cleft. Under conditions where Tyr411 is not acylated, the molar fraction of diazepam, diflunisal, ibuprofen, and 3-indoxyl-sulfate bound to HSA is higher than 0.9 whereas the molar fraction of propofol bound to HSA is ca. 0.5.     

Effect of Poor Access to Water and Sanitation As Risk Factors for Soil-Transmitted Helminth Infection: Selectiveness by the Infective Route

BackgroundSoil-transmitted helminth (STH) infections are a public health problem in resource-limited settings worldwide. Chronic STH infection impairs optimum learning and productivity, contributing to the perpetuation of the poverty-disease cycle. Regular massive drug administration (MDA) is the cardinal recommendation for its control; along with water, sanitation and hygiene (WASH) interventions. The impact of joint WASH interventions on STH infections has been reported; studies on the independent effect of WASH components are needed to contribute with the improvement of current recommendations for the control of STH. The aim of this study is to assess the association of lacking access to water and sanitation with STH infections, taking into account the differences in route of infection among species and the availability of adequate water and sanitation at home.ConclusionsLack of safe water and proper sanitation pose a risk of STH infections that is distinct according to the route of entry to the human host used by each of the STH species. Interventions aimed to improve water and sanitation access should be highlighted in the recommendations for the control of STH.     

Histamine Transmission Modulates the Phenotype of Murine Narcolepsy Caused by Orexin Neuron Deficiency

Narcolepsy type 1 is associated with loss of orexin neurons, sleep-wake derangements, cataplexy, and a wide spectrum of alterations in other physiological functions, including energy balance, cardiovascular, and respiratory control. It is unclear which narcolepsy signs are directly related to the lack of orexin neurons or are instead modulated by dysfunction of other neurotransmitter systems physiologically controlled by orexin neurons, such as the histamine system. To address this question, we tested whether some of narcolepsy signs would be detected in mice lacking histamine signaling (HDC-KO). Moreover, we studied double-mutant mice lacking both histamine signaling and orexin neurons (DM) to evaluate whether the absence of histamine signaling would modulate narcolepsy symptoms produced by orexin deficiency. Mice were instrumented with electrodes for recording the electroencephalogram and electromyogram and a telemetric arterial pressure transducer. Sleep attacks fragmenting wakefulness, cataplexy, excess rapid-eye-movement sleep (R) during the activity period, and enhanced increase of arterial pressure during R, which are hallmarks of narcolepsy in mice, did not occur in HDC-KO, whereas they were observed in DM mice. Thus, these narcolepsy signs are neither caused nor abrogated by the absence of histamine. Conversely, the lack of histamine produced obesity in HDC-KO and to a greater extent also in DM. Moreover, the regularity of breath duration during R was significantly increased in either HDC-KO or DM relative to that in congenic wild-type mice. Defects of histamine transmission may thus modulate the metabolic and respiratory phenotype of murine narcolepsy.     

Llama Nanoantibodies with Therapeutic Potential against Human Norovirus Diarrhea

Noroviruses are a major cause of acute gastroenteritis, but no vaccines or therapeutic drugs are available. Llama-derived single chain antibody fragments (also called VHH) are small, recombinant monoclonal antibodies of 15 kDa with several advantages over conventional antibodies. The aim of this study was to generate recombinant monoclonal VHH specific for the two major norovirus (NoV) genogroups (GI and GII) in order to investigate their potential as immunotherapy for the treatment of NoV diarrhea. To accomplish this objective, two llamas were immunized with either GI.1 (Norwalk-1968) or GII.4 (MD2004) VLPs. After immunization, peripheral blood lymphocytes were collected and used to generate two VHH libraries. Using phage display technology, 10 VHH clones specific for GI.1, and 8 specific for GII.4 were selected for further characterization. All VHH recognized conformational epitopes in the P domain of the immunizing VP1 capsid protein, with the exception of one GII.4 VHH that recognized a linear P domain epitope. The GI.1 VHHs were highly specific for the immunizing GI.1 genotype, with only one VHH cross-reacting with GI.3 genotype. The GII.4 VHHs reacted with the immunizing GII.4 strain and showed a varying reactivity profile among different GII genotypes. One VHH specific for GI.1 and three specific for GII.4 could block the binding of homologous VLPs to synthetic HBGA carbohydrates, saliva, and pig gastric mucin, and in addition, could inhibit the hemagglutination of red blood cells by homologous VLPs. The ability of Nov-specific VHHs to perform well in these surrogate neutralization assays supports their further development as immunotherapy for NoV treatment and immunoprophylaxis.     

Tetrahydrohyperforin Inhibits the Proteolytic Processing of Amyloid Precursor Protein and Enhances Its Degradation by Atg5-Dependent Autophagy

Alzheimer''s disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid-β (Aβ) peptide. We have previously shown that the compound tetrahydrohyperforin (IDN5706) prevents accumulation of Aβ species in an in vivo model of AD, however the mechanism that explains this reduction is not well understood. We show herein that IDN5706 decreases the levels of ER degradation enhancer, mannosidase alpha-like 1 (EDEM1), a key chaperone related to endoplasmic-reticulum-associated degradation (ERAD). Moreover, we observed that low levels of EDEM1 correlated with a strong activation of autophagy, suggesting a crosstalk between these two pathways. We observed that IDN5706 perturbs the glycosylation and proteolytic processing of the amyloid precursor protein (APP), resulting in the accumulation of immature APP (iAPP) in the endoplasmic reticulum. To investigate the contribution of autophagy, we tested the effect of IDN5706 in Atg5-depleted cells. We found that depletion of Atg5 enhanced the accumulation of iAPP in response to IDN5706 by slowing down its degradation. Our findings reveal that IDN5706 promotes degradation of iAPP via the activation of Atg5-dependent autophagy, shedding light on the mechanism that may contribute to the reduction of Aβ production in vivo.     

Diverging temperature response of tree stem CO2 release under dry and wet season conditions in a tropical montane moist forest

It is commonly presumed that plant respiratory CO₂ release increases with increasing temperature. However, we report on very contrasting stem CO₂ release (R _S)–temperature relationships of trees in a species-rich tropical montane forest of southern Ecuador under dry and wet season conditions. Rates of R _S were low and completely uncoupled from the dial temperature regime during the humid season. In contrast, during the dry season, R _S was generally higher and temperature sensitivity of R _S differed greatly in degree and even in the direction of response, indicating that temperature might not be the only determinant of R _S. In order to explain the heterogeneity of R _S, we related R _S to vapour pressure deficit, wind speed and solar radiation as important abiotic drivers influencing transpiration and photosynthesis. Stepwise multiple regression analyses with these meteorological predictors either were biased by high collinearity of the independent variables or could not enhance the ability to explain the variability of R _S. We assume maintenance respiration to dominate under humid conditions unfavourable for energy acquisition of the tree, thus explaining the pronounced uncoupling of R _S from atmospheric parameters. In contrast, the drier and hotter climate of the dry season seems to favour R _S via enhanced assimilatory substrate delivery and stem respiratory activity as well as elevated xylem sap CO₂ imports with increased transpiration. In addition, tree individual differences in the temperature responses of R _S may mirror diverging climatic adaptations of co-existing moist forest tree species which have their distribution centre either at higher or lower elevations.     

Molecular mechanisms involved in the enhancement of mitochondrial malate dehydrogenase activity by calcitriol in chick intestine

Mitochondrial malate dehydrogenase (mMDH) from the intestine is the NAD-linked oxidoreductase of the tricarboxylic acid cycle with the highest activity and response to vitamin D treatment in vitamin D-deficient chicks (?D). The aim of this study was to elucidate potential molecular mechanisms by which cholecalciferol or calcitriol enhances the activity of this enzyme. One group of animals used was composed of ?D and ?D treated with cholecalciferol or with calcitriol. A second group consisted of ?D and ?D supplemented with high Ca²⁺ diet. A third group included chicks receiving either a normal or a low Ca²⁺ diet. In some experiments, animals were injected with cycloheximide. Data showed that either vitamin D (cholecalciferol or calcitriol) or a low Ca²⁺ diet increases mMDH activity. High Ca²⁺ diet did not modify the intestinal mMDH activity from ?D. The mMDH activity from ?D remained unaltered when duodenal cells were exposed to 10^?8 mol/L calcitriol for 15 min. The enhancement of mMDH activity by calcitriol was completely abolished by simultaneous cycloheximide injection to ?D. mMDH mRNA levels, detected by RT-PCR, indicate that calcitriol did not affect gene expression. In contrast, Western blots show that calcitriol enhanced the protein expression. In conclusion, calcitriol stimulates intestinal mMDH activity by increasing protein synthesis. No response of mMDH activity by rapid effects of calcitriol or activation through increment of serum Ca²⁺ was demonstrated. Consequently, ATP production would be increased, facilitating the Ca²⁺ exit from the enterocytes via the Ca²⁺-ATPase and Na⁺/Ca²⁺ exchanger, which participate in the intestinal Ca²⁺ absorption.