Repression of a Potassium Channel by Nuclear Hormone Receptor and TGF-β Signaling Modulates Insulin Signaling in Caenorhabditis elegans

Transforming growth factor β (TGF-β) signaling acts through Smad proteins to play fundamental roles in cell proliferation, differentiation, apoptosis, and metabolism. The Receptor associated Smads (R-Smads) interact with DNA and other nuclear proteins to regulate target gene transcription. Here, we demonstrate that the Caenorhabditis elegans R-Smad DAF-8 partners with the nuclear hormone receptor NHR-69, a C. elegans ortholog of mammalian hepatocyte nuclear factor 4α HNF4α), to repress the exp-2 potassium channel gene and increase insulin secretion. We find that NHR-69 associates with DAF-8 both in vivo and in vitro. Functionally, daf-8 nhr-69 double mutants show defects in neuropeptide secretion and phenotypes consistent with reduced insulin signaling such as increased expression of the sod-3 and gst-10 genes and a longer life span. Expression of the exp-2 gene, encoding a voltage-gated potassium channel, is synergistically increased in daf-8 nhr-69 mutants compared to single mutants and wild-type worms. In turn, exp-2 acts selectively in the ASI neurons to repress the secretion of the insulin-like peptide DAF-28. Importantly, exp-2 mutation shortens the long life span of daf-8 nhr-69 double mutants, demonstrating that exp-2 is required downstream of DAF-8 and NHR-69. Finally, animals over-expressing NHR-69 specifically in DAF-28–secreting ASI neurons exhibit a lethargic, hypoglycemic phenotype that is rescued by exogenous glucose. We propose a model whereby DAF-8/R-Smad and NHR-69 negatively regulate the transcription of exp-2 to promote neuronal DAF-28 secretion, thus demonstrating a physiological crosstalk between TGF-β and HNF4α-like signaling in C. elegans. NHR-69 and DAF-8 dependent regulation of exp-2 and DAF-28 also provides a novel molecular mechanism that contributes to the previously recognized link between insulin and TGF-β signaling in C. elegans

Gene expression profiling of oxidative stress response of C. elegans aging defective AMPK mutants using massively parallel transcriptome sequencing

<p>Abstract</p> <p>Background</p> <p>A strong association between stress resistance and longevity in multicellular organisms has been established as many mutations that extend lifespan also show increased resistance to stress. AAK-2, the <it>C. elegans </it>homolog of an alpha subunit of AMP-activated protein kinase (AMPK) is an intracellular fuel sensor that regulates cellular energy homeostasis and functions in stress resistance and lifespan extension.</p> <p>Findings</p> <p>Here, we investigated global transcriptional responses of <it>aak-2 </it>mutants to oxidative stress and in turn identified potential downstream targets of AAK-2 involved in stress resistance in <it>C. elegans</it>. We employed massively parallel Illumina sequencing technology and performed comprehensive comparative transcriptome analysis. Specifically, we compared the transcriptomes of <it>aak-2 </it>and wild type animals under normal conditions and conditions of induced oxidative stress. This research has presented a snapshot of genome-wide transcriptional activities that take place in <it>C. elegans </it>in response to oxidative stress both in the presence and absence of AAK-2.</p> <p>Conclusions</p> <p>The analysis presented in this study has enabled us to identify potential genes involved in stress resistance that may be either directly or indirectly under the control of AAK-2. Furthermore, we have extended our current knowledge of general defense responses of <it>C. elegans </it>against oxidative stress supporting the function for AAK-2 in inhibition of biosynthetic processes, especially lipid synthesis, under oxidative stress and transcriptional regulation of genes involved in reproductive processes.</p

Electric Field-Driven Water Dipoles: Nanoscale Architecture of Electroporation

<div><p>Electroporation is the formation of permeabilizing structures in the cell membrane under the influence of an externally imposed electric field. The resulting increased permeability of the membrane enables a wide range of biological applications, including the delivery of normally excluded substances into cells. While electroporation is used extensively in biology, biotechnology, and medicine, its molecular mechanism is not well understood. This lack of knowledge limits the ability to control and fine-tune the process. In this article we propose a novel molecular mechanism for the electroporation of a lipid bilayer based on energetics analysis. Using molecular dynamics simulations we demonstrate that pore formation is driven by the reorganization of the interfacial water molecules. Our energetics analysis and comparisons of simulations with and without the lipid bilayer show that the process of poration is driven by field-induced reorganization of water dipoles at the water-lipid or water-vacuum interfaces into more energetically favorable configurations, with their molecular dipoles oriented in the external field. Although the contributing role of water in electroporation has been noted previously, here we propose that interfacial water molecules are the main players in the process, its initiators and drivers. The role of the lipid layer, to a first-order approximation, is then reduced to a relatively passive barrier. This new view of electroporation simplifies the study of the problem, and opens up new opportunities in both theoretical modeling of the process and experimental research to better control or to use it in new, innovative ways.</p></div

Enhanced greenhouse gas emission from exposed sediments along a hydroelectric reservoir during an extreme drought event

An active debate has been underway on the magnitude and duration of carbon (C) emissions from hydroelectric reservoirs, yet little attention has been paid to stochastic C emissions from reservoir sediments during extreme climatic events. A rare opportunity for field measurements of CO2 efflux from a hydroelectric reservoir in Korea during an extreme drought event was used to examine how prolonged droughts can affect microbial organic matter processing and the release of CO2, CH4 and N2O from exposed sediments. Chamber measurements of CO2 efflux along an exposed sediment transect, combined with high-frequency continuous sensor measurements of the partial pressure of CO2 (pCO(2)) in the reservoir surface water, exhibited extraordinary pulses of CO2 from exposed sediments and the turbulent inflowing water in contrast to a small CO2 sink in the main water body of the reservoir and a low efflux of CO2 from the flooded sediment. Significant increases in the production of CO2, CH4 and N2O observed in a laboratory incubation of sediments, together with enhanced activities of phenol oxidase and three hydrolases, indicate a temporary activation of microbial organic matter processing in the drying sediment. The results suggest that drought-triggered pulses of greenhouse gas emission from exposed sediments can offset the C accumulation in reservoir sediments over time scales of years to decades, reversing the trend of declining C emissions from aging reservoirsope

Divergent evolution of extreme production of variant plant monounsaturated fatty acids

Metabolic extremes provide opportunities to understand enzymatic and metabolic plasticity and biotechnological tools for novel biomaterial production. We discovered that seed oils of many Thunbergia species contain up to 92% of the unusual monounsaturated petroselinic acid (18:1Δ6), one of the highest reported levels for a single fatty acid in plants. Supporting the biosynthetic origin of petroselinic acid, we identified a Δ6-stearoyl-acyl carrier protein (18:0-ACP) desaturase from Thunbergia laurifolia, closely related to a previously identified Δ6-palmitoyl-ACP desaturase that produces sapienic acid (16:1Δ6)- rich oils in Thunbergia alata seeds. Guided by a T. laurifolia desaturase crystal structure obtained in this study, enzyme mutagenesis identified key amino acids for functional divergence of Δ6 desaturases from the archetypal Δ9-18:0-ACP desaturase and mutations that result in nonnative enzyme regiospecificity. Furthermore, we demonstrate the utility of the T. laurifolia desaturase for the production of unusual monounsaturated fatty acids in engineered plant and bacterial hosts. Through stepwise metabolic engineering, we provide evidence that divergent evolution of extreme petroselinic acid and sapienic acid production arises from biosynthetic and metabolic functional specialization and enhanced expression of specific enzymes to accommodate metabolism of atypical substrates

Associations of Dietary Intakes of Total and Specific Types of Fat with Blood Lipid Levels in the Filipino Women’s Diet and Health Study (FiLWHEL)

Background: Limited evidence exists on the association between dietary fat intake and lipid profiles in Southeast Asian populations. Objectives: We aimed to examine the cross-sectional associations of dietary intake of total and specific types of fat with dyslipidemia in Filipino immigrant women in Korea. Methods: We included 406 Filipino women married to Korean in the Filipino Women’s Diet and Health Study (FiLWHEL). Dietary fat intake was assessed using 24-hour recalls. Impaired blood lipid profiles were defined as high total cholesterol (TC) (≥200 mg/dL), high triglyceride (TG) (≥150 mg/dL), high LDL Cholesterol (LDL-C) (≥ 130 mg/dL), or low HDL cholesterol (HDL-C) (<50 mg/dL). The genomic DNA samples were genotyped using DNA chip. The odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using multivariate logistic regression. Results: Substituting carbohydrates with dietary saturated fat (SFA) intake was associated with increased prevalence of dyslipidemia; ORs (95% CIs) for subsequent tertiles compared to the first tertile were 2.28 (1.19–4.35), and 2.88 (1.29–6.39) (P for trend = 0.02). When we examined individual markers, ORs (95% CIs, P for trend) comparing the third to the first tertile were 3.62 (1.53–8.55, 0.01) for high TC, 1.46 (0.42–5.10, 0.72) for high TG, 4.00 (1.48–10.79, 0.02) for high LDL-C, and 0.69 (0.30–1.59, 0.36) for low HDL-C. When we examined the interaction by LDL-C-related polymorphisms, the association with dyslipidemia was more pronounced among participants with CC alleles than among those with T alleles of rs6102059 (P for interaction = 0.01). Conclusions: High dietary SFA intake was significantly associated with a high prevalence of dyslipidemia in Filipino women in Korea. Further prospective cohort studies are warranted to determine risk factors for CVD in Southeast Asian populations

Quantification of G i -Mediated Inhibition of Adenylyl Cyclase Activity Reveals That UDP Is a Potent Agonist of the Human P2Y 14 Receptor

ABSTRACT The P2Y 14 receptor was initially identified as a G protein-coupled receptor activated by UDP-glucose and other nucleotide sugars. We have developed several cell lines that stably express the human P2Y 14 receptor, allowing facile examination of its coupling to native G i family G proteins and their associated downstream signaling pathways (J Pharmacol Exp Ther 330: [162][163][164][165][166][167][168] 2009). In the current study, we examined P2Y 14 receptor-dependent inhibition of cyclic AMP accumulation in human embryonic kidney (HEK) 293, C6 glioma, and Chinese hamster ovary (CHO) cells stably expressing this receptor. Not only was the human P2Y 14 receptor activated by UDP-glucose, but it also was activated by UDP. The apparent efficacies of UDP and UDP-glucose were similar, and the EC 50 values (74, 33, and 29 nM) for UDP-dependent activation of the P2Y 14 receptor in HEK293, CHO, and C6 glioma cells, respectively, were similar to the EC 50 values (323, 132, and 72 nM) observed for UDP-glucose. UDP and UDP-glucose also stimulated extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in P2Y 14 receptor-expressing HEK293 cells but not in wild-type HEK293 cells. A series of analogs of UDP were potent P2Y 14 receptor agonists, but the naturally occurring nucleoside diphosphates, CDP, GDP, and ADP exhibited agonist potencies over 100-fold less than that observed with UDP. Two UDP analogs were identified that selectively activate the P2Y 14 receptor over the UDP-activated P2Y 6 receptor, and these molecules stimulated phosphorylation of ERK1/2 in differentiated human HL-60 promyeloleukemia cells, which natively express the P2Y 14 receptor but had no effect in wild-type HL-60 cells, which do not express the receptor. We conclude that UDP is an important cognate agonist of the human P2Y 14 receptor. The metabotropic P2Y receptors include a subgroup of five receptors, the P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , and P2Y 11 receptors, that primarily signal through G q -activated signaling pathways and a subgroup of three receptors, the P2Y 12 , P2Y 13 , and P2Y 14 receptors, that primarily signal by activating heterotrimeric G proteins of the G i famil

Molecular recognition in the P2Y14 receptor: Probing the structurally permissive terminal sugar moiety of uridine-5′-diphosphoglucose

The P2Y14 receptor, a nucleotide signaling protein, is activated by uridine-5′-diphosphoglucose 1 and other uracil nucleotides. We have determined that the glucose moiety of 1 is the most structurally permissive region for designing analogues of this P2Y14 agonist. For example, the carboxylate group of uridine-5′-diphosphoglucuronic acid proved to be suitable for flexible substitution by chain extension through an amide linkage. Functionalized congeners containing terminal 2-acylaminoethylamides prepared by this stratgegy retained P2Y14 activity, and molecular modeling predicted close proximity of this chain to the 2nd extracellular loop of the receptor. In addition, replacement of glucose with other sugars did not diminish P2Y14 potency. For example, the [5″]ribose derivative had an EC50 of 0.24 μM. Selective monofluorination of the glucose moiety indicated a role for the 2″- and 6″-hydroxyl groups of 1 in receptor recognition. The β-glucoside was 2-fold less potent than the native α-isomer, but methylene replacement of the 1″-oxygen abolished activity. Replacement of the ribose ring system with cyclopentyl or rigid bicyclo[3.1.0]hexane groups abolished activity. Uridine-5′-diphosphoglucose also activates the P2Y2 receptor, but the 2-thio analogue and several of the potent modified-glucose analogues were P2Y14-selective

A variant of green fluorescent protein exclusively deposited to active intracellular inclusion bodies

Background: Inclusion bodies (IBs) were generally considered to be inactive protein deposits and did not hold any attractive values in biotechnological applications. Recently, some IBs of recombinant proteins were confirmed to show their functional properties such as enzyme activities, fluorescence, etc. Such biologically active IBs are not commonly formed, but they have great potentials in the fields of biocatalysis, material science and nanotechnology. Results: In this study, we characterized the IBs of DL4, a deletion variant of green fluorescent protein which forms active intracellular aggregates. The DL4 proteins expressed in Escherichia coli were exclusively deposited to IBs, and the IBs were estimated to be mostly composed of active proteins. The spectral properties and quantum yield of the DL4 variant in the active IBs were almost same with those of its native protein. Refolding and stability studies revealed that the deletion mutation in DL4 didn&apos;t affect the folding efficiency of the protein, but destabilized its structure. Analyses specific for amyloid-like structures informed that the inner architecture of DL4 IBs might be amorphous rather than well-organized. The diameter of fluorescent DL4 IBs could be decreased up to 100-200 nm by reducing the expression time of the protein in vivo. Conclusions: To our knowledge, DL4 is the first GFP variant that folds correctly but aggregates exclusively in vivo without any self-aggregating/assembling tags. The fluorescent DL4 IBs have potentials to be used as fluorescent biomaterials. This study also suggests that biologically active IBs can be achieved through engineering a target protein itself.open0