Pathogen-responsive expression of a putative ATP-binding cassette transporter gene conferring resistance to the diterpenoid sclareol is regulated by multiple defense signaling pathways in arabidopsis

The ATP-binding cassette (ABC) transporters are encoded by large gene families in plants. Although these proteins are potentially involved in a number of diverse plant processes, currently, very little is known about their actual functions. In this paper, through a cDNA microarray screening of anonymous cDNA clones from a subtractive library, we identified an Arabidopsis gene (AtPDR12) putatively encoding a member of the pleiotropic drug resistance (PDR) subfamily of ABC transporters. AtPDR12 displayed distinct induction profiles after inoculation of plants with compatible and incompatible fungal pathogens and treatments with salicylic acid, ethylene, or methyl jasmonate. Analysis of AtPDR12 expression in a number of Arabidopsis defense signaling mutants further revealed that salicylic acid accumulation, NPR1. function, and sensitivity to jasmonates and ethylene were all required for pathogen-responsive expression of AtPDR12. Germination assays using seeds from an AtPDR12 insertion line in the presence of sclareol resulted in lower germination rates and much stronger inhibition of root elongation in the AtPDR12 insertion line than in wild-type plants. These results suggest that AtPDR12 may be functionally related to the previously identified ABC transporters SpTUR2 and NpABC1, which transport sclareol. Our data also point to a potential role for terpenoids in the Arabidopsis defensive armory.

The chicken ovalbumin upstream promoter-transcription factors modulate genes and pathways involved in skeletal muscle cell metabolism

The chicken ovalbumin upstream promoter-transcription factors ( COUP-TFs) are orphan members of the nuclear hormone receptor ( NR) superfamily. COUP-TFs are involved in organogenesis and neurogenesis. However, their role in skeletal muscle ( and other major mass tissues) and metabolism remains obscure. Skeletal muscle accounts for similar to 40% of total body mass and energy expenditure. Moreover, this peripheral tissue is a primary site of glucose and fatty acid utilization. We utilize small interfering RNA ( siRNA)-mediated attenuation of Coup-TfI and II ( mRNA and protein) in a skeletal muscle cell culture model to understand the regulatory role of Coup-Tfs in this energy demanding tissue. This targeted NR repression resulted in the significant attenuation of genes that regulate lipid mobilization and utilization ( including Ppar alpha, Fabp3, and Cpt-1). This was coupled to reduced fatty acid beta-oxidation. Additionally we observed significant attenuation of Ucp1, a gene involved in energy expenditure. Concordantly, we observed a 5-fold increase in ATP levels in cells with siRNA-mediated repression of Coup-TfI and II. Furthermore, the expression of classical liver X receptor ( LXR) target genes involved in reverse cholesterol transport ( Abca1 and Abcg1) were both significantly repressed. Moreover, we observed that repression of the Coup-Tfs ablated the activation of Abca1, and Abcg1 mRNA expression by the selective LXR agonist, T0901317. In concordance, Coup-Tf-siRNA-transfected cells were refractory to Lxr-mediated reduction of total intracellular cholesterol levels in contrast to the negative control cells. In agreement Lxr-mediated activation of the Abca1 promoter in Coup-Tf-siRNA cells was attenuated. Collectively, these data suggest a pivotal role for Coup-Tfs in the regulation of lipid utilization/cholesterol homeostasis in skeletal muscle cells and the modulation of Lxr-dependent gene regulation.

Investigation of the absorption pathways of some poorly absorbed drugs using human intestinal (CAC0-2) cell monolayers

Absorption across the gastro-intestinal epithelium is via two pathways; the transcellular and paracellular pathway. Caco-2 cells, when cultured on polycarbonate filters, formed a confluent monolayer with many properties of differentiated intestinal epithelial cells, As a model of human gastro-intestinaJ tract epithelia they were used to elucidate and characterise the transepithelial transport of two protein kinase C inhibitors, N-(3-chlorophenyl)-4-[2-(3-hydroxypropylamino)-4-pyridyl]-2-pyrimidinamin (CHPP) and N-benzoyl-staurosporine (NBS), and the polypeptide, human calcitonin. Lanthanum ions are proposed as a paracellular pathway inhibitor and tested with D-mannitol permeability and transepithelial electrical resistance measurements. The effect La3+ has on the carrier-mediated transport of D-glucose and Sodium taurocholate as well as the vesicularly transcytosed horseradish peroxidase was also investigated. As expected, 2 mM apical La3+ increases transepithelial electrical resistance 1.S-fold and decreases mannitol permeability by 63.0 % ± 1.37 %. This inhibition was not repeated by other cations. Apical 2 mM La3+ was found to decrease carrier-mediated D-glucose and taurocholate permeability by only 8.7 % ± 1.6 %, 26.3 % ± 5.0 %. There was no inhibitory effect on testosterone or PEG 4000 permeability observed with La3+. However, for horseradish peroxidase and human calcitonin permeability was decreased by 98.7 % ± 11.7%, and 96.2 % ± 0.8 % respectively by 2 mM La3+. Indicating that human calcitonin could also be transported by vesicular transcytosis. The addition of 2 mM La3+ to the apical surface of Caco-2 monolayers produces a paracellular pathway inhibition. Therefore, La3+ could be a useful additional tool in delineating the transepithelial pathway of passive drug absorption.

Adrenoceptors and intestinal fluid and electrolyte transport in the rat

Noradrenaline was found to significantly stimulate fluid and Na absorption across everted sacs of rat jejunum. Of a number of a1, and 2-adrenoceptor antagonists tested only prazosin significantly inhibited the stimulant effect of noradrenaline and further experiments revealed an antiabsorptive effect of prazosin alone. Theophylline reduced jejunal fluid and Na absorption and this effect was not reversed by 2-adrenoceptor stimulation in contrast to previous findings in vivo. Evidence suggests the everted sac preparation is not appropriate to the study of intestinal fluid and electrolyte transport. The investigation of Jejunal ion transport in vitro was continued using an Ussing chamber preparation. Selective 2-adrenoceptor stimulation was found to depress electrogenic anion secretion, as neurotoxin tetrodotoxin indicated that this was a direct epithelial effect. 2-adrenoceptor agonists have considerable therapeutic value as antisecretory agents and the model of rat jejunum in vitro represents a convenient experimental model for research in this area. The selective 2-adrenoceptor antagonist ICI 118551 decreased basal SCC and inhibited increases in SCC in response to isoprenaline or salbutamol indicating the presence of a 2-adrenoceptor mechanism mediating both secretory tone and increases in secretory processes. Many intestinal secretagogues elicit electrolyte secretion via the stimulation of intramural secretory nervous pathways. If these pathways involve the activation of 2-adrenoceptorsthe 2-adrenoceptor antagonists may be useful in the treatment of diarrhoeal diseases. A single pass lumen perfusion technique was used to investigate possible sympathetic tone over colonic fluid and electrolyte absorption in the rat colon in vivo. The technique employed appeared to lack the necessary resolution for this study and alternative approaches are discussed

Adrenoceptor influences on fluid and electrolyte transport in the rat intestine

An investigation of rat jejunal and distal colonic electrolyte transport in-vitro was undertaken using an Ussing chamber prepartion. Selective α2-adrenoceptor stimualtion in the jejunum was found to depress theo-phylline elevated anion secretion, as evidenced by decreases in short- circuit current (SCC). or α1 -Adrenoceptor stimulation, after α2 -adrenoceptor antagonism in the jejunum, evoked transient increases in basal anion secretion, as reflected by transient increases in basal SCC. The use of the neurotoxin tetrodotoxin indicated that this was a direct epithelial secretory effect. 5-hydroxytryptamine (5-HT) on the jejunum elicited transient increases in basal anion secretion, as demonstrated by transient increases in basal SCC. The use of tetrodotoxin, reserpine and α1 -adrenoceptor antagonists, indicated that a major component of this epithelial secretory effect by 5-HT, was associated with activation of intramural nervous pathways of the sympathetic nervous system, ultimately stimulating α1-adrenoceptors. This might represent an important secretory mechanism by 5-HT in the jejunum. β2-Adrenoceptor stimulation in the distal colon was found to decrease basal SCC, as evidenced by the metoprolol resistant effect of the selective β2- adrenoceptor agonist salbutamol, and lack of effect of the selective β1-adrenoceptor agonist prenalterol. An investigation of rat distal colonic fluid and electrolyte transport in-vivo was undertaken using an colonic loop technique. Although a basal colonic absorption of Na+ and Cl-, and a secretion of K+ were observed, these processes were not under tonic α-adrenergic regulation, as evidenced by the lack of effect of selective α-adrenoceptor antagonism. The secretory effects of prostaglandin-E2 were inhibited by α-adrenoceptor activation, whereas such stimulation did not evoke pro-absorptive responses upon basal transport, unlike noradrenaline.

Extra-nuclear telomerase reverse transcriptase (TERT) regulates glucose transport in skeletal muscle cells

Telomerase reverse transcriptase (TERT) is a key component of the telomerase complex. By lengthening telomeres in DNA strands, TERT increases senescent cell lifespan. Mice that lack TERT age much faster and exhibit age-related conditions such as osteoporosis, diabetes and neurodegeneration. Accelerated telomere shortening in both human and animal models has been documented in conditions associated with insulin resistance, including T2DM. We investigated the role of TERT, in regulating cellular glucose utilisation by using the myoblastoma cell line C2C12, as well as primary mouse and human skeletal muscle cells. Inhibition of TERT expression or activity by using siRNA (100. nM) or specific inhibitors (100. nM) reduced basal 2-deoxyglucose uptake by ~. 50%, in all cell types, without altering insulin responsiveness. In contrast, TERT over-expression increased glucose uptake by 3.25-fold. In C2C12 cells TERT protein was mostly localised intracellularly and stimulation of cells with insulin induced translocation to the plasma membrane. Furthermore, co-immunoprecipitation experiments in C2C12 cells showed that TERT was constitutively associated with glucose transporters (GLUTs) 1, 4 and 12 via an insulin insensitive interaction that also did not require intact PI3-K and mTOR pathways. Collectively, these findings identified a novel extra-nuclear function of TERT that regulates an insulin-insensitive pathway involved in glucose uptake in human and mouse skeletal muscle cells. © 2014 Elsevier B.V.

Geochemical determination of the fate and transport of injected fresh wastewater to a deep saline aquifer

Deep well injection into non-potable saline aquifers of treated domestic wastewater has been used in Florida for decades as a safe and effective alternative to ocean outfall disposal. The objectives of this study were to determine the fate and transport of injected wastewater at two deep well injection sites in Miami Dade County, Florida, USA. Detection of ammonium in the Middle Confining units of the Floridan aquifer above the injection zone at both sites has been interpreted as evidence of upward migration of injected wastewater, posing a risk to underground sources of drinking water. Historical water quality data, including ammonia, chloride, temperature, and pH from existing monitoring wells at both sites from 1983 to 2008, major ions collected monthly from 2006 and 2008, and a synoptic sampling event for stable isotopes, tritium, and dissolved gases in 2008, were used to determine the source of ammonium in groundwater and possible migration pathways. Geochemical modeling was used to determine possible effects of injected wastewater on native water and aquifer matrix geochemistry. Injected wastewater was determined to be the source of elevated ammonium concentrations above ambient water levels, based on the results of major ion concentrations, tritium, dissolved noble gases and 15N isotopes analyses. Various possible fluid migration pathways were identified at the sites. Data for the south site suggest buoyancy-driven vertical pathways to overlying aquifers bypassing the confining units, with little mixing of injected wastewater with native water as it migrated upward. Once it is introduced into an aquifer, the injectate appeared to migrate advectively with the regional groundwater flow. Geochemical modeling indicated that CO 2-enriched injected wastewater allowed for carbonate dissolution along the vertical pathways, enhancing permeability along these flowpaths. At the north site, diffusive upward flow through the confining units or offsite vertical pathways were determined to be possible, however no evidence was detected for any on-site confining unit bypass pathway. No evidence was observed at either site of injected wastewater migration to the Upper Floridan aquifer, which is used as a municipal water supply and for aquifer storage and recovery.

Arsenic transport by zebrafish aquaglyceroporins

Background Arsenic is one of the most ubiquitous toxins and endangers the health of tens of millions of humans worldwide. It is a mainly a water-borne contaminant. Inorganic trivalent arsenic (AsIII) is one of the major species that exists environmentally. The transport of AsIII has been studied in microbes, plants and mammals. Members of the aquaglyceroporin family have been shown to actively conduct AsIII and its organic metabolite, monomethylarsenite (MAsIII). However, the transport of AsIII and MAsIII in in any fish species has not been characterized. Results In this study, five members of the aquaglyceroporin family from zebrafish (Danio rerio) were cloned, and their ability to transport water, glycerol, and trivalent arsenicals (AsIII and MAsIII) and antimonite (SbIII) was investigated. Genes for at least seven aquaglyceroporins have been annotated in the zebrafish genome project. Here, five genes which are close homologues to human AQP3, AQP9 and AQP10 were cloned from a zebrafish cDNA preparation. These genes were namedaqp3, aqp3l, aqp9a, aqp9b and aqp10 according to their similarities to the corresponding human AQPs. Expression of aqp9a, aqp9b, aqp3, aqp3l and aqp10 in multiple zebrafish organs were examined by RT-PCR. Our results demonstrated that these aquaglyceroporins exhibited different tissue expression. They are all detected in more than one tissue. The ability of these five aquaglyceroporins to transport water, glycerol and the metalloids arsenic and antimony was examined following expression in oocytes from Xenopus leavis. Each of these channels showed substantial glycerol transport at equivalent rates. These aquaglyceroporins also facilitate uptake of inorganic AsIII, MAsIII and SbIII. Arsenic accumulation in fish larvae and in different tissues from adult zebrafish was studied following short-term arsenic exposure. The results showed that liver is the major organ of arsenic accumulation; other tissues such as gill, eye, heart, intestine muscle and skin also exhibited significant ability to accumulate arsenic. The zebrafish larvae also accumulate considerable amounts of arsenic. Conclusion This is the first molecular identification of fish arsenite transport systems and we propose that the extensive expression of the fish aquaglyceroporins and their ability to transport metalloids suggests that aquaglyceroporins are the major pathways for arsenic accumulation in a variety of zebrafish tissues. Uptake is one important step of arsenic metabolism. Our results will contribute to a new understanding of aquatic arsenic metabolism and will support the use of zebrafish as a new model system to study arsenic associated human diseases.

EFFECTS OF BUBBLE-FACILITATED VOC TRANSPORT FROM LNAPL SMEAR ZONES ON VAPOR INTRUSION

Light non-aqueous phase liquid (LNAPL) sources can pose a significant threat to indoor air through vapour intrusion (VI). Most conceptual and numerical models of VI assume that the transport of volatile organic compounds (VOCs) is a diffusion-limited process. Recently, alternate conditions have been identified that could lead to faster transport, including the presence of preferential pathways and methanogenic gas production. In this study, an additional mechanism that could lead to faster transport was investigated: bubble-facilitated VOC transport from LNAPL smear zones. A laboratory investigation was preformed using pentane in one-dimensional laboratory columns and two-dimensional visualization experiments. Results of the column experiments showed that average VOC mass fluxes in the bubble-facilitated columns were over two orders of magnitude greater than in the diffusion-limited columns. In addition, the flux signal was intermittent, consistent with expectations of bubble-facilitated transport as bubbles expand, mobilize and are released to the vadose zone at various times during the test. The results from the visualization experiments showed gas fingers growing and mobilizing over time, which supports the findings of the column experiments. In conclusion, these results demonstrate the potential for bubble-facilitated VOC transport to affect mass transfer in LNAPL smear zones, and lead to increased indoor air concentrations by VI.

De Novo Assembly And Transcriptome Analysis Of The Rubber Tree (hevea Brasiliensis) And Snp Markers Development For Rubber Biosynthesis Pathways.

Hevea brasiliensis (Willd. Ex Adr. Juss.) Muell.-Arg. is the primary source of natural rubber that is native to the Amazon rainforest. The singular properties of natural rubber make it superior to and competitive with synthetic rubber for use in several applications. Here, we performed RNA sequencing (RNA-seq) of H. brasiliensis bark on the Illumina GAIIx platform, which generated 179,326,804 raw reads on the Illumina GAIIx platform. A total of 50,384 contigs that were over 400 bp in size were obtained and subjected to further analyses. A similarity search against the non-redundant (nr) protein database returned 32,018 (63%) positive BLASTx hits. The transcriptome analysis was annotated using the clusters of orthologous groups (COG), gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Pfam databases. A search for putative molecular marker was performed to identify simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs). In total, 17,927 SSRs and 404,114 SNPs were detected. Finally, we selected sequences that were identified as belonging to the mevalonate (MVA) and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways, which are involved in rubber biosynthesis, to validate the SNP markers. A total of 78 SNPs were validated in 36 genotypes of H. brasiliensis. This new dataset represents a powerful information source for rubber tree bark genes and will be an important tool for the development of microsatellites and SNP markers for use in future genetic analyses such as genetic linkage mapping, quantitative trait loci identification, investigations of linkage disequilibrium and marker-assisted selection.

Ki-1/57 And Cgi-55 Ectopic Expression Impact Cellular Pathways Involved In Proliferation And Stress Response Regulation.

Ki-1/57 (HABP4) and CGI-55 (SERBP1) are regulatory proteins and paralogs with 40.7% amino acid sequence identity and 67.4% similarity. Functionally, they have been implicated in the regulation of gene expression on both the transcriptional and mRNA metabolism levels. A link with tumorigenesis is suggested, since both paralogs show altered expression levels in tumor cells and the Ki-1/57 gene is found in a region of chromosome 9q that represents a haplotype for familiar colon cancer. However, the target genes regulated by Ki-1/57 and CGI-55 are unknown. Here, we analyzed the alterations of the global transcriptome profile after Ki-1/57 or CGI-55 overexpression in HEK293T cells by DNA microchip technology. We were able to identify 363 or 190 down-regulated and 50 or 27 up-regulated genes for Ki-1/57 and CGI-55, respectively, of which 20 were shared between both proteins. Expression levels of selected genes were confirmed by qRT-PCR both after protein overexpression and siRNA knockdown. The majority of the genes with altered expression were associated to proliferation, apoptosis and cell cycle control processes, prompting us to further explore these contexts experimentally. We observed that overexpression of Ki-1/57 or CGI-55 results in reduced cell proliferation, mainly due to a G1 phase arrest, whereas siRNA knockdown of CGI-55 caused an increase in proliferation. In the case of Ki-1/57 overexpression, we found protection from apoptosis after treatment with the ER-stress inducer thapsigargin. Together, our data give important new insights that may help to explain these proteins putative involvement in tumorigenic events.

Improvement Of The Physical Performance Is Associated With Activation Of No/pgc-1α/mttfa Signaling Pathway And Increased Protein Expressions Of Electron Transport Chain In Gastrocnemius Muscle From Rats Supplemented With L-arginine.

To examine the influence of l-arginine supplementation in combination with physical training on mitochondrial biomarkers from gastrocnemius muscle and its relationship with physical performance. Male Wistar rats were divided into four groups: control sedentary (SD), sedentary supplemented with l-arginine (SDLA), trained (TR) and trained supplemented with l-arginine (TRLA). Supplementation of l-arginine was administered by gavage (62.5mg/ml/day/rat). Physical training consisted of 60min/day, 5days/week, 0% grade, speed of 1.2km/h. The study lasted 8weeks. Skeletal muscle mitochondrial enriched fraction as well as cytoplasmic fractions were obtained for Western blotting and biochemical analyses. Protein expressions of transcriptor coactivator (PGC-1α), transcriptor factors (mtTFA), ATP synthase subunit c, cytochrome oxidase (COXIV), constitutive nitric oxide synthases (eNOS and nNOS), Cu/Zn-superoxide dismutase (SOD) and manganese-SOD (Mn-SOD) were evaluated. We also assessed in plasma: lipid profile, glycemia and malondialdehyde (MDA) levels. The nitrite/nitrate (NOx(-)) levels were measured in both plasma and cytosol fraction of the gastrocnemius muscle. 8-week l-arginine supplementation associated with physical training was effective in promoting greater tolerance to exercise that was accompanied by up-regulation of the protein expressions of mtTFA, PGC-1α, ATP synthase subunit c, COXIV, Cu/Zn-SOD and Mn-SOD. The upstream pathway was associated with improvement of NO bioavailability, but not in NO production since no changes in nNOS or eNOS protein expressions were observed. This combination would be an alternative approach for preventing cardiometabolic diseases given that in overt diseases a profound impairment in the physical performance of the patients is observed.

Oropouche Virus Infection And Pathogenesis Is Restricted By Mavs, Irf-3, Irf-7, And Type I Ifn Signaling Pathways In Non-myeloid Cells.

Oropouche virus (OROV) is a member of the Orthobunyavirus genus in the Bunyaviridae family and a prominent cause of insect-transmitted viral disease in Central and South America. Despite its clinical relevance, little is known about OROV pathogenesis. To define the host defense pathways that control OROV infection and disease, we evaluated OROV pathogenesis and immune responses in primary cells and mice that were deficient in the RIG-I-like receptor signaling pathway (MDA5, RIG-I, or MAVS), downstream regulatory transcription factors (IRF-3 or IRF-7), IFN-β, or the receptor for type I IFN signaling (IFNAR). OROV replicated to higher levels in primary fibroblasts and dendritic cells lacking MAVS signaling, the transcription factors IRF-3 and IRF-7, or IFNAR. In mice, deletion of IFNAR, MAVS, or IRF-3 and IRF-7 resulted in uncontrolled OROV replication, hypercytokinemia, extensive liver damage, and death whereas wild-type (WT) congenic animals failed to develop disease. Unexpectedly, mice with a selective deletion of IFNAR on myeloid cells (CD11c Cre(+) Ifnar(f/f) or LysM Cre(+) Ifnar(f/f)) did not sustain enhanced disease with OROV or La Crosse virus, a closely related encephalitic orthobunyavirus. In bone marrow chimera studies, recipient irradiated Ifnar(-/-) mice reconstituted with WT hematopoietic cells sustained high levels of OROV replication and liver damage, whereas WT mice reconstituted with Ifnar(-/-) bone marrow were resistant to disease. Collectively, these results establish a dominant protective role for MAVS, IRF-3 and IRF-7, and IFNAR in restricting OROV virus infection and tissue injury, and suggest that IFN signaling in non-myeloid cells contributes to the host defense against orthobunyaviruses. Oropouche virus (OROV) is an emerging arthropod-transmitted orthobunyavirus that causes episodic outbreaks of a debilitating febrile illness in humans in countries of South and Central America. The continued expansion of the range and number of its arthropod vectors increases the likelihood that OROV will spread into new regions. At present, the pathogenesis of OROV in humans or other vertebrate animals remains poorly understood. To define cellular mechanisms of control of OROV infection, we performed infection studies in a series of primary cells and mice that were deficient in key innate immune genes involved in pathogen recognition and control. Our results establish that a MAVS-dependent type I IFN signaling pathway has a dominant role in restricting OROV infection and pathogenesis in vivo.

A Leucine-rich Diet Modulates The Tumor-induced Down-regulation Of The Mapk/erk And Pi3k/akt/mtor Signaling Pathways And Maintains The Expression Of The Ubiquitin-proteasome Pathway In The Placental Tissue Of Nmri Mice.

Placental tissue injury is concomitant with tumor development. We investigated tumor-driven placental damage by tracing certain steps of the protein synthesis and degradation pathways under leucine-rich diet supplementation in MAC16 tumor-bearing mice. Cell signaling and ubiquitin-proteasome pathways were assessed in the placental tissues of pregnant mice, which were distributed into three groups on a control diet (pregnant control, tumor-bearing pregnant, and pregnant injected with MAC-ascitic fluid) and three other groups on a leucine-rich diet (pregnant, tumor-bearing pregnant, and pregnant injected with MAC-ascitic fluid). MAC tumor growth down-regulated the cell-signaling pathways of the placental tissue and decreased the levels of IRS-1, Akt/PKB, Erk/MAPK, mTOR, p70S6K, STAT3, and STAT6 phosphorylated proteins, as assessed by the multiplex Millipore Luminex assay. Leucine supplementation maintained the levels of these proteins within the established cell-signaling pathways. In the tumor-bearing group (MAC) only, the placental tissue showed increased PC5 mRNA expression, as assessed by quantitative RT-PCR, decreased 19S and 20S protein expression, as assessed by Western blot analysis, and decreased placental tyrosine levels, likely reflecting up-regulation of the ubiquitin-proteasome pathway. Similar effects were found in the pregnant injected with MAC-ascitic fluid group, confirming that the effects of the tumor were mimicked by MAC-ascitic fluid injection. Although tumor progression occurred, the degradation pathway-related protein levels were modulated under leucine-supplementation conditions. In conclusion, tumor evolution reduced the protein expression of the cell-signaling pathway associated with elevated protein degradation, thereby jeopardizing placental activity. Under the leucine-rich diet, the impact of cancer on placental function could be minimized by improving the cell-signaling activity and reducing the proteolytic process.

Characterisation Of The Membrane Transport Of Pilocarpine In Cell Suspension Cultures Of Pilocarpus Microphyllus.

Pilocarpine is an alkaloid obtained from the leaves of Pilocarpus genus, with important pharmaceutical applications. Previous reports have investigated the production of pilocarpine by Pilocarpus microphyllus cell cultures and tried to establish the alkaloid biosynthetic route. However, the site of pilocarpine accumulation inside of the cell and its exchange to the medium culture is still unknown. Therefore, the aim of this study was to determine the intracellular accumulation of pilocarpine and characterise its transport across membranes in cell suspension cultures of P. microphyllus. Histochemical analysis and toxicity assays indicated that pilocarpine is most likely stored in the vacuoles probably to avoid cell toxicity. Assays with exogenous pilocarpine supplementation to the culture medium showed that the alkaloid is promptly uptaken but it is rapidly metabolised. Treatment with specific ABC protein transporter inhibitors and substances that disturb the activity of secondary active transporters suppressed pilocarpine uptake and release suggesting that both proteins may participate in the traffic of pilocarpine to inside and outside of the cells. As bafilomicin A1, a specific V-type ATPase inhibitor, had little effect and NH4Cl (induces membrane proton gradient dissipation) had moderate effect, while cyclosporin A and nifedipine (ABC proteins inhibitors) strongly inhibited the transport of pilocarpine, it is believed that ABC proteins play a major role in the alkaloid transport across membranes but it is not the exclusive one. Kinetic studies supported these results.