S-adenosylmethionine and Pneumocystis carinii

We previously reported that S-adenosylmethionine (AdoMet), a key molecule in methylation reactions and polyamine biosynthesis, enhances axenic culture of the AIDS-associated opportunistic fungal pathogen Pneumocystis carinii. Here we report that AdoMet is absolutely required for continuous growth. Two transporters are present, one high affinity, K(m) = 4.5 microm, and one low affinity, K(m) = 333 microm. The physiologically relevant high affinity transporter has a pH optimum of 7.5 and no related natural compounds compete for uptake. Transport is 98% inhibited at 4 degrees C, 24% inhibited by 20 mm sodium azide, and 95% inhibited by the combination of 20 mm sodium azide and 1 mm salicylhydroxamic acid; thus transport is active and dependent on both a cytochrome chain and an alternative oxidase. In vitro, AdoMet is used at a rate of 1. 40 x 10(7) molecules cell(-1) min(-1). AdoMet synthetase activity was not detected by a sensitive radiolabel incorporation assay capable of detecting 0.1% of the activity in rat liver. In addition, the AdoMet plasma concentration of rats is inversely correlated with the number of P. carinii in the lungs. These findings demonstrate that P. carinii is an AdoMet auxotroph. The uptake and metabolism of this compound are rational chemotherapeutic targets.     

Effects of plant biomass, plant diversity, and water content on bacterial communities in soil lysimeters: implications for the determinants of bacterial diversity

Soils may comprise tens of thousands to millions of bacterial species. It is still unclear whether this high level of diversity is governed by functional redundancy or by a multitude of ecological niches. In order to address this question, we analyzed the reproducibility of bacterial community composition after different experimental manipulations. Soil lysimeters were planted with four different types of plant communities, and the water content was adjusted. Group-specific phylogenetic fingerprinting by PCR-denaturing gradient gel electrophoresis revealed clear differences in the composition of Alphaproteobacteria, Betaproteobacteria, Bacteroidetes, Chloroflexi, Planctomycetes, and Verrucomicrobia populations in soils without plants compared to that of populations in planted soils, whereas no influence of plant species composition on bacterial diversity could be discerned. These results indicate that the presence of higher plant species affects the species composition of bacterial groups in a reproducible manner and even outside of the rhizosphere. In contrast, the environmental factors tested did not affect the composition of Acidobacteria, Actinobacteria, Archaea, and Firmicutes populations. One-third (52 out of 160) of the sequence types were found to be specifically and reproducibly associated with the absence or presence of plants. Unexpectedly, this was also true for numerous minor constituents of the soil bacterial assemblage. Subsequently, one of the low-abundance phylotypes (beta10) was selected for studying the interdependence under particular experimental conditions and the underlying causes in more detail. This so-far-uncultured phylotype of the Betaproteobacteria species represented up to 0.18% of all bacterial cells in planted lysimeters compared to 0.017% in unplanted systems. A cultured representative of this phylotype exhibited high physiological flexibility and was capable of utilizing major constituents of root exudates. Our results suggest that the bacterial species composition in soil is determined to a significant extent by abiotic and biotic factors, rather than by mere chance, thereby reflecting a multitude of distinct ecological niches.     

Novel biomarkers to assess in utero effects of maternal opioid use: First steps toward understanding short‐ and long‐term neurodevelopmental sequelae

Maternal opioid use disorder is common, resulting in significant neonatal morbidity and cost. Currently, it is not possible to predict which opioid‐exposed newborns will require pharmacotherapy for neonatal abstinence syndrome. Further, little is known regarding the effects of maternal opioid use disorder on the developing human brain. We hypothesized that novel methodologies utilizing fetal central nervous system‐derived extracellular vesicles isolated from maternal blood can address these gaps in knowledge. Plasma from opioid users and controls between 9 and 21 weeks was precipitated and extracellular vesicles were isolated. Mu opioid and cannabinoid receptor levels were quantified. Label‐free proteomics studies and unbiased small RNA next generation sequencing was performed in paired fetal brain tissue. Maternal opioid use disorder increased mu opioid receptor protein levels in extracellular vesicles independent of opioid equivalent dose. Moreover, cannabinoid receptor levels in extracellular vesicles were upregulated with opioid exposure indicating cross talk with endocannabinoids. Maternal opioid use disorder was associated with significant changes in extracellular vesicle protein cargo and fetal brain micro RNA expression, especially in male fetuses. Many of the altered cargo molecules and micro RNAs identified are associated with adverse clinical neurodevelopmental outcomes. Our data suggest that assays relying on extracellular vesicles isolated from maternal blood extracellular vesicles may provide information regarding fetal response to opioids in the setting of maternal opioid use disorder. Prospective clinical studies are needed to evaluate the association between extracellular vesicle biomarkers, risk of neonatal abstinence syndrome and neurodevelopmental outcomes.     

Changes in Protein Expression in Two Cholangiocarcinoma Cell Lines Undergoing Formation of Multicellular Tumor Spheroids In Vitro

Epithelial-to-Mesenchymal Transition (EMT) is relevant in malignant growth and frequently correlates with worsening disease progression due to its implications in metastases and resistance to therapeutic interventions. Although EMT is known to occur in several types of solid tumors, the information concerning tumors arising from the epithelia of the bile tract is still limited. In order to approach the problem of EMT in cholangiocarcinoma, we decided to investigate the changes in protein expression occurring in two cell lines under conditions leading to growth as adherent monolayers or to formation of multicellular tumor spheroids (MCTS), which are considered culture models that better mimic the growth characteristics of in-vivo solid tumors. In our system, changes in phenotypes occur with only a decrease in transmembrane E-cadherin and vimentin expression, minor changes in the transglutaminase protein/activity but with significant differences in the proteome profiles, with declining and increasing expression in 6 and in 16 proteins identified by mass spectrometry. The arising protein patterns were analyzed based on canonical pathways and network analysis. These results suggest that significant metabolic rearrangements occur during the conversion of cholangiocarcinomas cells to the MCTS phenotype, which most likely affect the carbohydrate metabolism, protein folding, cytoskeletal activity, and tissue sensitivity to oxygen.     

SILAC-Based Mass Spectrometry Analysis Reveals That Epibrassinolide Induces Apoptosis via Activating Endoplasmic Reticulum Stress in Prostate Cancer Cells

Epibrassinolide (EBR) is a polyhydroxylated sterol derivative and biologically active compound of the brassinosteroids. In addition to well-described roles in plant growth, EBR induces apoptosis in the LNCaP prostate cancer cells expressing functional androgen receptor (AR). Therefore, it is suggested that EBR might have an inhibitory potential on androgen receptor signaling pathway. However, the mechanism by which EBR exerts its effects on LNCaP is poorly understood. To address this gap in knowledge, we used an unbiased global proteomics approach, i.e., stable-isotope labeling by amino acids in cell culture (SILAC). In total, 964 unique proteins were identified, 160 of which were differentially expressed after 12 h of EBR treatment. The quantification of the differentially expressed proteins revealed that the expression of the unfolded protein response (UPR) chaperone protein, calreticulin (CALR), was dramatically downregulated. The decrease in CALR expression was also validated by immunoblotting. Because our data revealed the involvement of the UPR in response to EBR exposure, we evaluated the expression of the other UPR proteins. We demonstrated that EBR treatment downregulated calnexin and upregulated BiP and IRE1α expression levels and induced CHOP translocation from the cytoplasm to nucleus. The translocation of CHOP was associated with caspase-9 and caspase-3 activation after a 12 h EBR treatment. Co-treatment of EBR with rapamycin, an upstream mTOR pathway inhibitor, prevented EBR-induced cell viability loss and PARP cleavage in LNCaP prostate cancer cells, suggesting that EBR could induce ER stress in these cells. In addition, we observed similar results in DU145 cells with nonfunctional androgen receptor. When proteasomal degradation of proteins was blocked by MG132 co-treatment, EBR treatment further induced PARP cleavage relative to drug treatment alone. EBR also induced Ca²⁺ sequestration, which confirmed the alteration of the ER pathway due to drug treatment. Therefore, we suggest that EBR promotes ER stress and induces apoptosis.     

Evaluation of anxiolytic properties of Gotukola – (Centella asiatica) extracts and asiaticoside in rat behavioral models

The ayurvedic medicinal plant Gotukola (Centella asiatica) was evaluated for its anxiolytic properties. Specifically, this study assessed the effects of: Gotukola plant materials of different genotypic origin; hexane, ethyl acetate and methanol extracts of Gotukola; and asiaticoside, a triterpenic compound isolated from Gotukola. Various paradigms were used to assess the anxiolytic activity, including the elevated plus maze (EPM), open field, social interaction, locomotor activity, punished drinking (Vogel) and novel cage tests. The EPM test revealed that Gotukola, its methanol and ethyl acetate extracts as well as the pure asiaticoside, imparted anxiolytic activity. Furthermore, the asiaticoside did not affect locomotor activity, suggesting these compounds do not have sedative effects in rodents.     

Dimethyl carbonate as potential reactant in non-catalytic biodiesel production by supercritical method

In this study, the non-catalytic supercritical method has been studied in utilizing dimethyl carbonate. It was demonstrated that, the supercritical dimethyl carbonate process without any catalysts applied, converted triglycerides to fatty acid methyl esters with glycerol carbonate and citramalic acid as by-products, while free fatty acids were converted to fatty acid methyl esters with glyoxal. After 12 min of reaction at 350 degrees C/20 MPa, rapeseed oil treated with supercritical dimethyl carbonate reached 94% (w/w) yield of fatty acid methyl ester. The by-products from this process which are glycerol carbonate and citramalic acid are much higher in value than glycerol produced by the conventional process. In addition, the yield of the fatty acid methyl esters as biodiesel was almost at par with supercritical methanol method. Therefore, supercritical dimethyl carbonate process can be a good candidate as an alternative biodiesel production process.