Glutathione transferase mu 2 protects glioblastoma cells against aminochrome toxicity by preventing autophagy and lysosome dysfunction

U373MG cells constitutively express glutathione S-transferase mu 2 (GSTM2) and exhibit ³H-dopamine uptake, which is inhibited by 2 µM of nomifensine and 15 µM of estradiol. We generated a stable cell line (U373MGsiGST6) expressing an siRNA against GSTM2 that resulted in low GSTM2 expression (26% of wild-type U373MG cells). A significant increase in cell death was observed when U373MGsiGST6 cells were incubated with 50 µM purified aminochrome (18-fold increase) compared with wild-type cells. The incubation of U373MGsiGST6 cells with 75 µM aminochrome resulted in the formation of autophagic vacuoles containing undigested cellular components, as determined using transmission electron microscopy. A significant increase in autophagosomes was determined by measuring endogenous LC3-II, a significant decrease in cell death was observed in the presence of bafilomycin A₁, and a significant increase in cell death was observed in the presence of trehalose. A significant increase in LAMP2 immunostaining was observed, a significant decrease in bright red fluorescence of lysosomes with acridine orange was observed, and bafilomycin A₁ pretreatment reduced the loss of lysosome acidity. A significant increase in cell death was observed in the presence of lysosomal protease inhibitors. Aggregation of TUBA/α-tubulin (tubulin, α) and SQSTM1 protein accumulation were also observed. Moreover, a significant increase in the number of lipids droplets was observed compared with U373MG cells with normal expression of GSTM2. These results support the notion that GSTM2 is a protective enzyme against aminochrome toxicity in astrocytes and that aminochrome cell death in U373MGsiGST6 cells involves autophagic-lysosomal dysfunction.     

A Systems Biology Approach Investigating the Effect of Probiotics on the Vaginal Microbiome and Host Responses in a Double Blind,Placebo-Controlled Clinical Trial of Post-Menopausal Women

A lactobacilli dominated microbiota in most pre and post-menopausal women is an indicator of vaginal health. The objective of this double blinded, placebo-controlled crossover study was to evaluate in 14 post-menopausal women with an intermediate Nugent score, the effect of 3 days of vaginal administration of probiotic L. rhamnosus GR-1 and L. reuteri RC-14 (2.5×10⁹ CFU each) on the microbiota and host response. The probiotic treatment did not result in an improved Nugent score when compared to when placebo. Analysis using 16S rRNA sequencing and metabolomics profiling revealed that the relative abundance of Lactobacillus was increased following probiotic administration as compared to placebo, which was weakly associated with an increase in lactate levels. A decrease in Atopobium was also observed. Analysis of host responses by microarray showed the probiotics had an immune-modulatory response including effects on pattern recognition receptors such as TLR2 while also affecting epithelial barrier function. This is the first study to use an interactomic approach for the study of vaginal probiotic administration in post-menopausal women. It shows that in some cases multifaceted approaches are required to detect the subtle molecular changes induced by the host to instillation of probiotic strains.

Trial Registration

ClinicalTrials.gov NCT02139839     

Butanol Isomers Exert Distinct Effects on Voltage-Gated Calcium Channel Currents and Thus Catecholamine Secretion in Adrenal Chromaffin Cells

Butanol (C₄H₁₀OH) has been used both to dissect the molecular targets of alcohols/general anesthetics and to implicate phospholipase D (PLD) signaling in a variety of cellular functions including neurotransmitter and hormone exocytosis. Like other primary alcohols, 1-butanol is a substrate for PLD and thereby disrupts formation of the intracellular signaling lipid phosphatidic acid. Because secondary and tertiary butanols do not undergo this transphosphatidylation, they have been used as controls for 1-butanol to implicate PLD signaling. Recently, selective pharmacological inhibitors of PLD have been developed and, in some cases, fail to block cellular functions previously ascribed to PLD using primary alcohols. For example, exocytosis of insulin and degranulation of mast cells are blocked by primary alcohols, but not by the PLD inhibitor FIPI. In this study we show that 1-butanol reduces catecholamine secretion from adrenal chromaffin cells to a much greater extent than tert-butanol, and that the PLD inhibitor VU0155056 has no effect. Using fluorescent imaging we show the effect of these drugs on depolarization-evoked calcium entry parallel those on secretion. Patch-clamp electrophysiology confirmed the peak amplitude of voltage-gated calcium channel currents (I_Ca) is inhibited by 1-butanol, with little or no block by secondary or tert-butanol. Detailed comparison shows for the first time that the different butanol isomers exert distinct, and sometimes opposing, effects on the voltage-dependence and gating kinetics of I_Ca. We discuss these data with regard to PLD signaling in cellular physiology and the molecular targets of general anesthetics.     

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1 hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.     

A modified method for determining protein binding capacity of plant polyphenolics using radiolabelled protein

A modified radiochemical protein binding method for determining the protein binding capacity of plant polyphenolics (tannins) is described. Purified tannin or unfractionated plant extracts were immobilised on filter paper discs and incubated with the 125I-labelled bovine serum albumin. Protein bound to the disc was proportional to the amount of tannin applied to the disc, although at high concentrations of polyphenolics the discs became saturated and the relationship was no longer applicable. The method was validated using purified procyanidin from Sorghum grain and has been applied to crude polyphenolic extracts from maple, white oak, black oak, walnut and tulip poplar leaves. Specific chemical assays for the determination of proanthocyanidins (acid butanol method) and hydrolysable tannins (modified potassium iodate method) were employed to validate the new protein binding method with the complex plant extracts.     

Control of Substrate Specificity by Active-Site Residues in Nitrobenzene Dioxygenase

Nitrobenzene 1,2-dioxygenase from Comamonas sp. strain JS765 catalyzes the initial reaction in nitrobenzene degradation, forming catechol and nitrite. The enzyme also oxidizes the aromatic rings of mono- and dinitrotoluenes at the nitro-substituted carbon, but the basis for this specificity is not understood. In this study, site-directed mutagenesis was used to modify the active site of nitrobenzene dioxygenase, and the contribution of specific residues in controlling substrate specificity and enzyme performance was evaluated. The activities of six mutant enzymes indicated that the residues at positions 258, 293, and 350 in the α subunit are important for determining regiospecificity with nitroarene substrates and enantiospecificity with naphthalene. The results provide an explanation for the characteristic specificity with nitroarene substrates. Based on the structure of nitrobenzene dioxygenase, substitution of valine for the asparagine at position 258 should eliminate a hydrogen bond between the substrate nitro group and the amino group of asparagine. Up to 99% of the mononitrotoluene oxidation products formed by the N258V mutant were nitrobenzyl alcohols rather than catechols, supporting the importance of this hydrogen bond in positioning substrates in the active site for ring oxidation. Similar results were obtained with an I350F mutant, where the formation of the hydrogen bond appeared to be prevented by steric interference. The specificity of enzymes with substitutions at position 293 varied depending on the residue present. Compared to the wild type, the F293Q mutant was 2.5 times faster at oxidizing 2,6-dinitrotoluene while retaining a similar K_m for the substrate based on product formation rates and whole-cell kinetics.     

The type III TGF-β receptor betaglycan transmembrane-cytoplasmic domain fragment is stable after ectodomain cleavage and is a substrate of the intramembrane protease γ-secretase

The Type III TGF-β receptor, betaglycan, is a widely expressed proteoglycan co-receptor for TGF-β superfamily ligands. The full-length protein undergoes ectodomain cleavage with release of a soluble ectodomain fragment. The fate of the resulting transmembrane-cytoplasmic fragment, however, has never been explored. We demonstrate here that the transmembrane-cytoplasmic fragment is stable in transfected cells and in cell lines expressing endogenous betaglycan. Production of this fragment is inhibited by the ectodomain shedding inhibitor TAPI-2. Treatment of cells with inhibitors of the intramembrane protease γ-secretase stabilizes this fragment, suggesting that it is a substrate of γ-secretase. Expression of the transmembrane-cytoplasmic fragment as well as γ-secretase inhibitor stabilization are independent of TGF-β1 or -β2 and are unaffected by mutation of the cytoplasmic domain serines that undergo phosphorylation. γ-Secretase inhibition or the expression of a transmembrane-cytoplasmic fragment in HepG2 cells blunted TGF-β2 signaling. Our findings thus suggest that the transmembrane-cytoplasmic fragment remaining after betaglycan ectodomain cleavage is stable and a substrate of γ-secretase, which may have significant implications for the TGF-β signaling response.     

Structural determinants for the ouabain-stimulated increase in Na–K ATPase activity

Recent studies suggest that at low concentrations, ouabain increases Na–K ATPase and NHE1 activity and activates the Src signaling cascade in proximal tubule cells. Our laboratory demonstrated that low concentrations of ouabain increase blood pressure in rats. We hypothesize that ouabain-induced increase in blood pressure and Na–K ATPase activity requires NHE1 activity and association. To test this hypothesis we treated rats with ouabain (1 μg kg body wt^− 1 day^− 1) for 9 days in the presence or absence of the NHE1 inhibitor, zoniporide. Ouabain stimulated a significant increase in blood pressure which was prevented by zoniporide. Using NHE1-expressing Human Kidney cells 2 (HK2), 8 (HK8) and 11 (HK11) and Mouse Kidney cells from Wild type (WT) and NHE1 knock-out mice (SWE) cell lines, we show that ouabain stimulated Na–K ATPase activity and surface expression in a Src-dependent manner in NHE1-expressing cells but not in NHE1-deplete cells. Zoniporide prevented ouabain-induced stimulation of ⁸⁶Rb uptake in the NHE1-expressing cells. FRET and TIRF microscopy showed that ouabain increased association between GFP-NHE1 and mCherry-Na–K ATPase transfected into NHE1-deficient SWE cells. Mutational analysis demonstrated that the caveolin binding motif (CBM) of Na–K ATPase α1 is required for translocation of both Na–K ATPase α1 and NHE1 to the basolateral membrane. Mutations in activity or scaffold domains of NHE1 resulted in loss of ouabain-mediated regulation of Na–K ATPase. These results support that NHE1 is required for the ouabain-induced increase in blood pressure, and that the caveolin binding motif of Na–K ATPase α1 as well as the activity and scaffolding domains of NHE1 are required for their functional association.     

Fitness consequences of maternal and grandmaternal effects

Transgenerational effects are broader than only parental relationships. Despite mounting evidence that multigenerational effects alter phenotypic and life‐history traits, our understanding of how they combine to determine fitness is not well developed because of the added complexity necessary to study them. Here, we derive a quantitative genetic model of adaptation to an extraordinary new environment by an additive genetic component, phenotypic plasticity, maternal and grandmaternal effects. We show how, at equilibrium, negative maternal and negative grandmaternal effects maximize expected population mean fitness. We define negative transgenerational effects as those that have a negative effect on trait expression in the subsequent generation, that is, they slow, or potentially reverse, the expected evolutionary dynamic. When maternal effects are positive, negative grandmaternal effects are preferred. As expected under Mendelian inheritance, the grandmaternal effects have a lower impact on fitness than the maternal effects, but this dual inheritance model predicts a more complex relationship between maternal and grandmaternal effects to constrain phenotypic variance and so maximize expected population mean fitness in the offspring.     

The Chemical Diversity of Lantana camara: Analyses of Essential Oil Samples from Cuba,Nepal, and Yemen

The aerial parts of Lantana camara L. were collected from three different geographical locations: Artemisa (Cuba), Biratnagar (Nepal), and Sana'a (Yemen). The essential oils were obtained by hydrodistillation and analyzed by gas chromatography/mass spectrometry. A cluster analysis of 39 L. camara essential oil compositions revealed eight major chemotypes: β‐caryophyllene, germacrene D, ar‐curcumene/zingiberene, γ‐curcumen‐15‐al/epi‐β‐bisabolol, (E)‐nerolidol, davanone, eugenol/alloaromadendrene, and carvone. The sample from Cuba falls into the group dominated by (E)‐nerolidol, the sample from Nepal is a davanone chemotype, and the sample from Yemen belongs to the β‐caryophyllene chemotype. The chemical composition of L. camara oil plays a role in the biological activity; the β‐caryophyllene and (E)‐nerolidol chemotypes showed antimicrobial and cytotoxic activities.