Hypomethylation of DNA during differentiation of friend erythroleukemia cells

DNA from mammalian cells has been shown to contain significant amounts of 5-methyl cytosine resulting from enzymatic transfer of methyl groups from s-adenosylmethionine to cytosine residues in the DNA polymer. The function of this modification is not known. We have found that DNA synthesized during chemically induced differentiation of friend erythroleukemia cells is hypomethylated, as measured by its ability to accept methyl groups transferred by homologous DNA methyltransferases in vitro. The extent of hypomethylation detected by this sensitive method is small, a decrease of less than 1.6 percent in 5-methylcytosine content. Hypomethylated DNA can be isolated from friend erythroleukemia cells grown in the presence of dimethyl sulfoxide, butyrate, hexamethylene-bis- acetamide, pentamethylene-bis acetamide, and ethionine. However, hypomethylated DNA is found only under conditions where differentiation is actually induced. DNA isolated from cells of a dimethyl sulfoxide- resistant subclone grown in the presence of that agent is not hypomethylated, although DNA of these cells becomes hypomethylated after growth in the presence of inducers that can trigger their differentiation. We also find that the DNA of friend erythroleukemia cells does not become hypomethylated when the cells are exposed to inducing agents in the presence of substances that inhibit differentiation. These results suggest a close link between genome modification by methylation and differentiation of friend erythroleukemia cells.     

The effect of areneboronic acids on the alkaline conversion of d-glucose into d-fructose

Benzeneboronic acid, 4-methoxybenzeneboronic acid, 3-nitrobenzeneboronic acid, and sulphonated benzeneboronic acid have been used to displace the pseudo-equilibria established in aqueous alkali between d-glucose, d-fructose, and d-mannose to give greatly increased yields of d-fructose. The effect of reaction temperature, pH, overall concentration, and molar ratio of acid:sugar on the yield of d-fructose has been investigated by using an automated assay for d-fructose.     

Odorant-stimulated phosphoinositide signaling in mammalian olfactory receptor neurons

Recent evidence has revived interest in the idea that phosphoinositides (PIs) may play a role in signal transduction in mammalian olfactory receptor neurons (ORNs). To provide direct evidence that odorants indeed activate PI signaling in ORNs, we used adenoviral vectors carrying two different fluorescently tagged probes, the pleckstrin homology (PH) domains of phospholipase Cδ1 (PLCδ1) and the general receptor of phosphoinositides (GRP1), to monitor PI activity in the dendritic knobs of ORNs in vivo. Odorants mobilized PI(4,5)P₂/IP₃ and PI(3,4,5)P₃, the substrates and products of PLC and PI3K. We then measured odorant activation of PLC and PI3K in olfactory ciliary-enriched membranes in vitro using a phospholipid overlay assay and ELISAs. Odorants activated both PLC and PI3K in the olfactory cilia within 2 s of odorant stimulation. Odorant-dependent activation of PLC and PI3K in the olfactory epithelium could be blocked by enzyme-specific inhibitors. Odorants activated PLC and PI3K with partially overlapping specificity. These results provide direct evidence that odorants indeed activate PI signaling in mammalian ORNs in a manner that is consistent with the idea that PI signaling plays a role in olfactory transduction.     

Time series metagenomic sampling of the Thermopyles,Greece, geothermal springs reveals stable microbial communities dominated by novel sulfur-oxidizing chemoautotrophs

Geothermal springs are essentially unaffected by environmental conditions aboveground as they are continuously supplied with subsurface water with little variability in chemistry. Therefore, changes in their microbial community composition and function, especially over a long period, are expected to be limited but this assumption has not yet been rigorously tested. Toward closing this knowledge gap, we applied whole metagenome sequencing to 17 water samples collected between 2010 and 2016 from the Thermopyles sulfur-rich geothermal springs in central Greece. As revealed by 16S rRNA gene fragments recovered in the metagenomes, Epsilonproteobacteria-related operational taxonomic units (OTUs) dominated most samples and grouping of samples based on OTU abundances exhibited no apparent seasonal pattern. Similarities between samples regarding functional gene content were high, with all samples sharing >70% similarity in functional pathways. These community-wide patterns were further confirmed by analysis of metagenome-assembled genomes (MAGs), which showed that novel species and genera of the chemoautotrophic Campylobacterales order dominated the springs. These MAGs carried different pathways for thiosulfate or sulfide oxidation coupled to carbon fixation pathways. Overall, our study showed that even in the long term, functions of microbial communities in a moderately hot terrestrial spring remain stable, presumably driving the corresponding stability in community structure.     

Genome repository of oil systems: An interactive and searchable database that expands the catalogued diversity of crude oil-associated microbes

Microbial communities ultimately control the fate of petroleum hydrocarbons (PHCs) that enter the natural environment, but the interactions of microbes with PHCs and the environment are highly complex and poorly understood. Genome-resolved metagenomics can help unravel these complex interactions. However, the lack of a comprehensive database that integrates existing genomic/metagenomic data from oil environments with physicochemical parameters known to regulate the fate of PHCs currently limits data analysis and interpretations. Here, we curated a comprehensive, searchable database that documents microbial populations in natural oil ecosystems and oil spills, along with available underlying physicochemical data, geocoded via geographic information system to reveal their geographic distribution patterns. Analysis of the ~2000 metagenome-assembled genomes (MAGs) available in the database revealed strong ecological niche specialization within habitats. Over 95% of the recovered MAGs represented novel taxa underscoring the limited representation of cultured organisms from oil-contaminated and oil reservoir ecosystems. The majority of MAGs linked to oil-contaminated ecosystems were detectable in non-oiled samples from the Gulf of Mexico but not in comparable samples from elsewhere, indicating that the Gulf is primed for oil biodegradation. The repository should facilitate future work toward a predictive understanding of the microbial taxa and their activities that control the fate of oil spills.     

Biochemical Large-Scale Interaction Analysis of Murine Olfactory Receptors and Associated Signaling Proteins with Post-Synaptic Density 95, Drosophila Discs Large,Zona-Occludens 1 (PDZ) Domains

G protein-coupled receptors (GPCRs) constitute the largest family among mammalian membrane proteins and are capable of initiating numerous essential signaling cascades. Various GPCR-mediated pathways are organized into protein microdomains that can be orchestrated and regulated through scaffolding proteins, such as PSD-95/discs-large/ZO1 (PDZ) domain proteins. However, detailed binding characteristics of PDZ–GPCR interactions remain elusive because these interactions seem to be more complex than previously thought. To address this issue, we analyzed binding modalities using our established model system. This system includes the 13 individual PDZ domains of the multiple PDZ domain protein 1 (MUPP1; the largest PDZ protein), a broad range of murine olfactory receptors (a multifaceted gene cluster within the family of GPCRs), and associated olfactory signaling proteins. These proteins were analyzed in a large-scale peptide microarray approach and continuative interaction studies. As a result, we demonstrate that canonical binding motifs were not overrepresented among the interaction partners of MUPP1. Furthermore, C-terminal phosphorylation and distinct amino acid replacements abolished PDZ binding promiscuity. In addition to the described in vitro experiments, we identified new interaction partners within the murine olfactory epithelium using pull-down-based interactomics and could verify the partners through co-immunoprecipitation. In summary, the present study provides important insight into the complexity of the binding characteristics of PDZ–GPCR interactions based on olfactory signaling proteins, which could identify novel clinical targets for GPCR-associated diseases in the future.PDZ domain proteins comprise one of the largest families among interaction domain scaffolding proteins and are highly abundant in various multicellular eukaryotic species. These proteins fulfill important physiological functions in a broad range of different tissues and cells as they orchestrate complex protein networks. Among putative PDZ interaction partners, one important protein family is the group of GPCRs¹, constituting the largest family of membrane proteins in mammals (1). Here, signal efficiency, speed, desensitization, and internalization can be modulated by PDZ proteins (2–5). Olfactory receptors (ORs) represent a multigene family within this group of seven-transmembrane domain proteins and encompass 2% of the mammalian genome (6). Belonging to class I GPCRs, ORs share many general features of this receptor family, making them an interesting target for interactions involving PDZ proteins. Until recently, an organizing complex builder, such as the inactivation no afterpotential D (InaD) protein in the visual system of Drosophila melanogaster (7, 8), could not be clearly identified for olfactory signaling.The multiple PDZ domain protein 1, with 13 individual PDZ domains, represents the largest of the described PDZ proteins to date (9) and interacts with different GPCRs (10–12). One well-described example is its interaction with GABA_B receptors, leading to enhanced receptor stability at the plasma membrane and prolonged signaling duration (2). In previous studies, we demonstrated that PDZ domains 1 + 2 can interact with a selected subset of ORs (13). Furthermore, we showed that MUPP1 binds to a specific OR and that most of the described proteins are involved in mammalian olfactory signal transduction in the native system, making MUPP1 a promising candidate for orchestrating the olfactory system (14).Many PDZ–ligand interactions depend on classical binding motifs at the ligand''s C-terminal end, thereby building weak transient protein complexes (15, 16). However, an increasing number of PDZ interactions have emerged that seem to provide more complex binding modalities, differing from the canonical interactions (17, 18). Ligand binding seems not to be exclusively restricted to C-terminal sites, and PDZ domains cannot be distinctly classified but are evenly distributed throughout a selective space (17, 19–21). Therefore, it is of great interest to analyze OR–PDZ interactions to characterize the putative binding requirements and to further investigate the role of MUPP1 in olfactory signaling.In the present study, we characterized the binding modalities between the 13 individual PDZ domains of MUPP1 and a broad range of murine olfactory receptors in a large-scale approach, indicating that classical binding motifs were not overrepresented among the evaluated binding partners. In addition, we identified new binding partners from the murine olfactory epithelium using pull-down-based interactomics.     

Deep soil carbon dynamics are driven more by soil type than by climate: a worldwide meta‐analysis of radiocarbon profiles

The response of soil carbon dynamics to climate and land‐use change will affect both the future climate and the quality of ecosystems. Deep soil carbon (>20 cm) is the primary component of the soil carbon pool, but the dynamics of deep soil carbon remain poorly understood. Therefore, radiocarbon activity (C), which is a function of the age of carbon, may help to understand the rates of soil carbon biodegradation and stabilization. We analyzed the published C contents in 122 profiles of mineral soil that were well distributed in most of the large world biomes, except for the boreal zone. With a multivariate extension of a linear mixed‐effects model whose inference was based on the parallel combination of two algorithms, the expectation–maximization (EM) and the Metropolis–Hasting algorithms, we expressed soil C profiles as a four‐parameter function of depth. The four‐parameter model produced insightful predictions of soil C as dependent on depth, soil type, climate, vegetation, land‐use and date of sampling (). Further analysis with the model showed that the age of topsoil carbon was primarily affected by climate and cultivation. By contrast, the age of deep soil carbon was affected more by soil taxa than by climate and thus illustrated the strong dependence of soil carbon dynamics on other pedologic traits such as clay content and mineralogy.     

Activation of an Olfactory Receptor Inhibits Proliferation of Prostate Cancer Cells

Olfactory receptors (ORs) are expressed not only in the sensory neurons of the olfactory epithelium, where they detect volatile substances, but also in various other tissues where their potential functions are largely unknown. Here, we report the physiological characterization of human OR51E2, also named prostate-specific G-protein-coupled receptor (PSGR) due to its reported up-regulation in prostate cancer. We identified androstenone derivatives as ligands for the recombinant receptor. PSGR can also be activated with the odorant β-ionone. Activation of the endogenous receptor in prostate cancer cells by the identified ligands evoked an intracellular Ca²⁺ increase. Exposure to β-ionone resulted in the activation of members of the MAPK family and inhibition of cell proliferation. Our data give support to the hypothesis that because PSGR signaling could reduce growth of prostate cancer cells, specific receptor ligands might therefore be potential candidates for prostate cancer treatment.Excessive signaling by G-protein-coupled receptors (GPCRs)³ such as endothelin A receptor (1), bradykinin 1 receptor (2), follicle-stimulating hormone receptor (3), and thrombin receptor (4, 5) is known to occur in prostate cancers due to strong overexpression of the respective receptors. Activation of some of these GPCRs results in androgen-independent androgen receptor activation, thus promoting the transition of prostate cancer cells from an androgen-dependent to an androgen-independent state (6, 7).The prostate-specific G-protein-coupled receptor (PSGR) is a class A GPCR that was initially identified as a prostate-specific tumor biomarker (8–10). It is specifically expressed in prostate epithelial cells, and its expression increases significantly in human prostate intraepithelial neoplasia and prostate tumors, suggesting that PSGR may play an important role in early prostate cancer development and progression (9, 11). Although expression of the human PSGR was found to be prostate-specific (10, 12), mRNA can also be detected in the olfactory zone and the medulla oblongata of the human brain (12). Human PSGR shares 93% amino acid homology to the respective mouse and rat homologues, which are also expressed in the brain (12). Interestingly, PSGR has numerous sequence motifs in common with the large superfamily of olfactory receptors (ORs), which build the largest class of human GPCRs and allow the recognition of a wide range of structurally diverse molecules in the nasal epithelium (13–15). Recently, also the steroid hormones androstenone and androstadienone were identified as OR ligands (16). In addition to their role in the sensory neurons of the nose, ORs have been found in different tissues throughout the body (17, 18). Their function(s) in these extranasal locations are questionable except for in a few cases where functional studies have been performed in spermatozoa (19, 20) and in enterochromaffin cells of the gastrointestinal tract (21).Here, we report the identification of steroid ligands of heterologously expressed PSGR and investigate the functional relevance of PSGR expression in prostate tissue. Steroid hormones elicited rapid Ca²⁺ responses in the LNCaP prostate cancer cell line and in primary human prostate epithelial cells. Moreover, activated PSGR causes phosphorylation of p38 and stress-activated protein kinase/c-Jun NH₂-terminal kinase (SAPK/JNK) mitogen-activated protein kinases (MAPKs), resulting in reduced proliferation rates in prostate cancer cells.     

Odorant-Dependent Generation of Nitric Oxide in Mammalian Olfactory Sensory Neurons

The gaseous signalling molecule nitric oxide (NO) is involved in various physiological processes including regulation of blood pressure, immunocytotoxicity and neurotransmission. In the mammalian olfactory bulb (OB), NO plays a role in the formation of olfactory memory evoked by pheromones as well as conventional odorants. While NO generated by the neuronal isoform of NO synthase (nNOS) regulates neurogenesis in the olfactory epithelium, NO has not been implicated in olfactory signal transduction. We now show the expression and function of the endothelial isoform of NO synthase (eNOS) in mature olfactory sensory neurons (OSNs) of adult mice. Using NO-sensitive micro electrodes, we show that stimulation liberates NO from isolated wild-type OSNs, but not from OSNs of eNOS deficient mice. Integrated electrophysiological recordings (electro-olfactograms or EOGs) from the olfactory epithelium of these mice show that NO plays a significant role in modulating adaptation. Evidence for the presence of eNOS in mature mammalian OSNs and its involvement in odorant adaptation implicates NO as an important new element involved in olfactory signal transduction. As a diffusible messenger, NO could also have additional functions related to cross adaptation, regeneration, and maintenance of MOE homeostasis.