Diverse sulfate-reducing bacteria of the Desulfosarcina/Desulfococcus clade are the key alkane degraders at marine seeps

Biogeochemical and microbiological data indicate that the anaerobic oxidation of non-methane hydrocarbons by sulfate-reducing bacteria (SRB) has an important role in carbon and sulfur cycling at marine seeps. Yet, little is known about the bacterial hydrocarbon degraders active in situ. Here, we provide the link between previous biogeochemical measurements and the cultivation of degraders by direct identification of SRB responsible for butane and dodecane degradation in complex on-site microbiota. Two contrasting seep sediments from Mediterranean Amon mud volcano and Guaymas Basin (Gulf of California) were incubated with ¹³C-labeled butane or dodecane under sulfate-reducing conditions and analyzed via complementary stable isotope probing (SIP) techniques. Using DNA- and rRNA-SIP, we identified four specialized clades of alkane oxidizers within Desulfobacteraceae to be distinctively active in oxidation of short- and long-chain alkanes. All clades belong to the Desulfosarcina/Desulfococcus (DSS) clade, substantiating the crucial role of these bacteria in anaerobic hydrocarbon degradation at marine seeps. The identification of key enzymes of anaerobic alkane degradation, subsequent β-oxidation and the reverse Wood–Ljungdahl pathway for complete substrate oxidation by protein-SIP further corroborated the importance of the DSS clade and indicated that biochemical pathways, analog to those discovered in the laboratory, are of great relevance for natural settings. The high diversity within identified subclades together with their capability to initiate alkane degradation and growth within days to weeks after substrate amendment suggest an overlooked potential of marine benthic microbiota to react to natural changes in seepage, as well as to massive hydrocarbon input, for example, as encountered during anthropogenic oil spills.     

Synthesis by Ring‐Closing Alkyne Metathesis with Selective Hydrogenation,and Olfactory Comparison of (7E)‐ and (7Z)‐Cyclohexadec‐7‐enone (Aurelione®)

Both C?C‐bond isomers of cyclohexadec‐7‐enone ( 6 , Aurelione^®) were selectively synthesized via cyclohexadec‐7‐ynol ( 16 ) by ring‐closing alkyne metathesis of icosa‐2,18‐diyn‐9‐ol ( 15 ), employing an in situ‐formed catalyst from Mo(CO)₆ and 4‐(trifluoromethyl)phenol. Pyridinium chlorochromate (PCC) oxidation and subsequent Lindlar hydrogenation afforded the (7Z)‐configured isomer (7Z)‐ 6 , while hydrosilylation of the intermediate cyclohexadec‐7‐ynone ( 17 ), followed by desilylation, provided the (7E)‐configured cyclohexadec‐7‐enone ((7E)‐ 6 ). The substrate for the alkyne metathesis was prepared from cycloheptanone ( 7 ) by cycloaddition of chloromethylcarbene to its trimethylsilyl enol ether 8 , and subsequent ring enlargement of the adduct 9 under rearrangement to 2‐methylcyclooct‐2‐enone ( 10 ), which was subjected to Weitz? Scheffer epoxidation and Eschenmoser? Ohloff fragmentation to non‐7‐ynal ( 12 ). Its reaction with the Grignard reagent of 11‐bromoundec‐2‐yne ( 14 ), prepared from the corresponding alcohol 13 by Appel? Lee bromination, furnished the icosa‐2,18‐diyn‐9‐ol ( 15 ). While both isomers of cyclohexadec‐7‐enone ( 6 ) possess warm and powdery musk odors with tobacco‐type ambery accents, (7Z)‐ 6 is more animalic and waxy, whereas (7E)‐ 6 was found to be more floral, sweet, and hay‐like in tonality. Interestingly, however, with odor detection thresholds of 2.0 ng/l air and 2.3 ng/l air, respectively, both (7Z)‐ 6 and (7E)‐ 6 were found to be almost identical in their odor strength, with the (7Z)‐ 6 being only very slightly more powerful.     

Evaluation of ubiquitinated proteins by proteomics reveals the role of the ubiquitin proteasome system in the regulation of Grp75 and Grp78 chaperone proteins during intestinal inflammation

The ubiquitin proteasome system (UPS) is the major pathway of intracellular protein degradation and may be involved in the pathophysiology of inflammatory bowel diseases or irritable bowel syndrome. UPS specifically degrades proteins tagged with an ubiquitin chain. We aimed to identify polyubiquitinated proteins during inflammatory response in intestinal epithelial HCT‐8 cells by a proteomic approach. HCT‐8 cells were incubated with interleukin 1β, tumor necrosis factor‐α, and interferon‐γ for 2 h. Total cellular protein extracts were separated by 2D gel electrophoresis and analyzed by an immunodetection using antiubiquitin antibody. Differential ubiquitinated proteins were then identified by LC‐ESI MS/MS. Seven proteins were differentially ubiquitinated between control and inflammatory conditions. Three of them were chaperones: Grp75 and Hsc70 were more ubiquitinated (p < 0.05) and Grp78 was less ubiquitinated (p < 0.05) under inflammatory conditions. The results for Grp75 and Grp78 were then confirmed in HCT‐8 cells and in 2‐4‐6‐trinitrobenzen sulfonic acid induced colitis in rats mimicking inflammatory bowel disease by immunoprecipitation. No difference was observed in irritable bowel syndrome like model. In conclusion, we showed that a proteomic approach is suitable to identify ubiquitinated proteins and that UPS‐regulated expression of Grp75 and Grp78 may be involved in inflammatory response. Further studies should lead to the identification of ubiquitin ligases responsible for Grp75 and Grp78 ubiquitination.     

Natural and Phosphorothioate-Modified Oligodeoxyribonucleotides Exhibit a Non-Random Cellular Distribution

Abstract

Addition of specific antisense oligomers to LTK- cells infected with HSV-1 has been shown to decrease viral production (1,8). We have investigated the cellular components that contain these oligomers and a phosphorothioate derivative by Normarski light microscopy and gel analysis of sucrose gradient cell fractions. Fractionation analysis suggests that these oligomers are distributed throughout the cell in a non-random manner and gel analysis suggest that intact oligomers are not equally distributed in the cytosol, nuclear or membrane component. Information about the cellular location of antisense oligomers should aid in the understanding of their antiviral effect and in the design of more effective oligonucleotide derivatives as potential antiviral agents.     

Cardiac oxidative stress in a mouse model of neutral lipid storage disease

Cardiac oxidative stress has been implicated in the pathogenesis of hypertrophy, cardiomyopathy and heart failure. Systemic deletion of the gene encoding adipose triglyceride lipase (ATGL), the enzyme that catalyzes the rate-limiting step of triglyceride lipolysis, results in a phenotype characterized by severe steatotic cardiac dysfunction. The objective of the present study was to investigate a potential role of oxidative stress in cardiac ATGL deficiency. Hearts of mice with global ATGL knockout were compared to those of mice with cardiomyocyte-restricted overexpression of ATGL and to those of wildtype littermates. Our results demonstrate that oxidative stress, measured as lucigenin chemiluminescence, was increased ~ 6-fold in ATGL-deficient hearts. In parallel, cytosolic NADPH oxidase subunits p67phox and p47phox were upregulated 4–5-fold at the protein level. Moreover, a prominent upregulation of different inflammatory markers (tumor necrosis factor α, monocyte chemotactant protein-1, interleukin 6, and galectin-3) was observed in those hearts. Both the oxidative and inflammatory responses were abolished upon cardiomyocyte-restricted overexpression of ATGL. Investigating the effect of oxidative and inflammatory stress on nitric oxide/cGMP signal transduction we observed a ~ 2.5-fold upregulation of soluble guanylate cyclase activity and a ~ 2-fold increase in cardiac tetrahydrobiopterin levels. Systemic treatment of ATGL-deficient mice with the superoxide dismutase mimetic Mn(III)tetrakis (4-benzoic acid) porphyrin did not ameliorate but rather aggravated cardiac oxidative stress. Our data suggest that oxidative and inflammatory stress seems involved in lipotoxic heart disease. Upregulation of soluble guanylate cyclase and cardiac tetrahydrobiopterin might be regarded as counterregulatory mechanisms in cardiac ATGL deficiency.     

cAMP-stimulated phosphorylation of diaphanous 1 regulates protein stability and interaction with binding partners in adrenocortical cells

Diaphanous homologue 1 (DIAPH1) is a Rho effector protein that coordinates cellular dynamics by regulating microfilament and microtubule function. We previously showed that DIAPH1 plays an integral role in regulating the production of cortisol by controlling the rate of mitochondrial movement, by which activation of the adrenocorticotropin (ACTH)/cAMP signaling pathway stimulates mitochondrial trafficking and promotes the interaction between RhoA and DIAPH1. In the present study we use mass spectrometry to identify DIAPH1 binding partners and find that DIAPH1 interacts with several proteins, including RhoA, dynamin-1, kinesin, β-tubulin, β-actin, oxysterol-binding protein (OSBP)–related protein 2 (ORP2), and ORP10. Moreover, DIAPH1 is phosphorylated in response to dibutyryl cAMP (Bt₂cAMP) at Thr-759 via a pathway that requires extracellular signal-related kinase (ERK). Alanine substitution of Thr-759 renders DIAPH1 more stable and attenuates the interaction between DIAPH1 and kinesin, ORP2, and actin but has no effect on the ability of the protein to interact with RhoA or β-tubulin. Finally, overexpression of a DIAPH1 T759A mutant significantly decreases the rate of Bt₂cAMP-stimulated mitochondrial movement. Taken together, our findings establish a key role for phosphorylation in regulating the stability and function of DIAPH1.