Fermentation of methanethiol and dimethylsulfide by a newly isolated methanogenic bacterium

From dilution series in defined mineral medium, a marine iregular coccoid methanogenic bacterium (strain MTP4) was isolated that was able to grow on methanethiol as sole source of energy. The strain also grew on dimethylsulfide, mono-, di-, and trimethylamine, methanol and acetate. On formate the organism produced methane without significant growth. Optimal growth on MT, with doubling times of about 20 h, occurred at 30°C in marine medium. The isolate required p-aminobenzoate and a further not identified vitamin. Strain MTP4 had a high tolerance to hydrogen sulfide but was very sensitive to mechanical forces or addition of detergents such as Triton X-100 or sodium dodecylsulfate. Methanethiol was fermented by strain MTP4 according to the following equation:

Formation of dimethylsulfide and methanethiol from methoxylated aromatic compounds and inorganic sulfide by newly isolated anaerobic bacteria

Formation of gas and of methylated sulfur compounds was observed in anaerobic enrichment cultures with methoxylated aromatic compounds as substrates. Via direct dilution of mud samples in defined reduced media supplemented with trimethoxybenzoate or syringate two new strains of anaerobic homoacetogenic bacteria (strain TMBS4 and strain SA2) were obtained in pure culture. Both strains produced dimethylsulfide and methanethiol during growth on methoxylated aromatic compounds. Growth tests and determination of stoichiometries demonstrated that the volatile sulfur compounds were formed from the methyl group at the aromatic ring and the sulfide added as reducing agent to the medium (R = aromatic residue): 2 R - O - CH₃ + H₂ S 2 R - OH + (CH₃)₂SDimethylsulfide was the major organic sulfur compound formed, whereas methanethiol appeared only as intermediate in small quantities. The isolates grew also with trihydroxybenzenes such as gallate, phloroglucinol, or pyrogallol without formation of methylated sulfur compounds. The aromatic compounds were degraded to acetate. The freshwater strain TMBS4 also fermented pyruvate. Other aliphatic or aromatic compounds were not utilized. External electron acceptors (sulfate, nitrate, fumarate) were not reduced. Both strains were mesophilic and formed rod-shaped, non-motile, Gram-negative cells. Spore formation was not observed. Tentatively, both isolates can be affiliated to the genus Pelobacter.Abbreviations TMB 3,4,5-trimethoxybenzoate - MT methanethiol - DMS dimethylsulfide     

Murine erythrocyte ankyrin cDNA: highly conserved regions of the regulatory domain

Ankyrin is an essential link between cytoskeletal proteins, such as spectrin, and membrane bound proteins, such as protein 3, the erythrocyte anion exchanger. Although the amino acid structure of human ankyrin is known, the functional regions have been only partially defined. Sequence comparisons between mouse and human ankyrin offer one mechanism of identifying highly conserved regions that probably have functional significance. We report the isolation and sequencing of a series of overlapping murine erythroid ankyrin (Ank-1) cDNAs from spleen and reticulocyte libraries (total span 6238 bp) and identify potentially important regions of murine-human reticulocyte ankyrin homology. Comparison of the predicted peptide sequences of mouse and human erythroid ankyrins shows that these ankyrins are highly conserved in both the N-terminal, protein 3 binding domain (96% amino acid identity) and in the central spectrin-binding domain (97% identity), but differ in the C-terminal regulatory domain (79% identity). However, the C-terminal regulatory domain contains two regions of peptide sequence that are perfectly conserved. We postulate these regions are important in the regulatory functions of this domain.     

Bacterial Disproportionation of Elemental Sulfur Coupled to Chemical Reduction of Iron or Manganese

A new chemolithotrophic bacterial metabolism was discovered in anaerobic marine enrichment cultures. Cultures in defined medium with elemental sulfur (S⁰) and amorphous ferric hydroxide (FeOOH) as sole substrates showed intense formation of sulfate. Furthermore, precipitation of ferrous sulfide and pyrite was observed. The transformations were accompanied by growth of slightly curved, rod-shaped bacteria. The quantification of the products revealed that S⁰ was microbially disproportionated to sulfate and sulfide, as follows: 4S⁰ + 4H₂O → SO₄^2- + 3H₂S + 2H⁺. Subsequent chemical reactions between the formed sulfide and the added FeOOH led to the observed precipitation of iron sulfides. Sulfate and iron sulfides were also produced when FeOOH was replaced by FeCO₃. Further enrichment with manganese oxide, MnO₂, instead of FeOOH yielded stable cultures which formed sulfate during concomitant reduction of MnO₂ to Mn²⁺. Growth of small rod-shaped bacteria was observed. When incubated without MnO₂, the culture did not grow but produced small amounts of SO₄^2- and H₂S at a ratio of 1:3, indicating again a disproportionation of S⁰. The observed microbial disproportionation of S⁰ only proceeds significantly in the presence of sulfide-scavenging agents such as iron and manganese compounds. The population density of bacteria capable of S⁰ disproportionation in the presence of FeOOH or MnO₂ was high, > 10⁴ cm^-3 in coastal sediments. The metabolism offers an explanation for recent observations of anaerobic sulfide oxidation to sulfate in anoxic sediments.     

Complete genome sequence of Desulfocapsa sulfexigens,a marine deltaproteobacterium specialized in disproportionating inorganic sulfur compounds

Desulfocapsa sulfexigens SB164P1 (DSM 10523) belongs to the deltaproteobacterial family Desulfobulbaceae and is one of two validly described members of its genus. This strain was selected for genome sequencing, because it is the first marine bacterium reported to thrive on the disproportionation of elemental sulfur, a process with a unresolved enzymatic pathway in which elemental sulfur serves both as electron donor and electron acceptor. Furthermore, in contrast to its phylogenetically closest relatives, which are dissimilatory sulfate-reducers, D. sulfexigens is unable to grow by sulfate reduction and appears metabolically specialized in growing by disproportionating elemental sulfur, sulfite or thiosulfate with CO₂ as the sole carbon source. The genome of D. sulfexigens contains the set of genes that is required for nitrogen fixation. In an acetylene assay it could be shown that the strain reduces acetylene to ethylene, which is indicative for N-fixation. The circular chromosome of D. sulfexigens SB164P1 comprises 3,986,761 bp and harbors 3,551 protein-coding genes of which 78% have a predicted function based on auto-annotation. The chromosome furthermore encodes 46 tRNA genes and 3 rRNA operons.     

Rrp1b, a new candidate susceptibility gene for breast cancer progression and metastasis

A novel candidate metastasis modifier, ribosomal RNA processing 1 homolog B (Rrp1b), was identified through two independent approaches. First, yeast two-hybrid, immunoprecipitation, and functional assays demonstrated a physical and functional interaction between Rrp1b and the previous identified metastasis modifier Sipa1. In parallel, using mouse and human metastasis gene expression data it was observed that extracellular matrix (ECM) genes are common components of metastasis predictive signatures, suggesting that ECM genes are either important markers or causal factors in metastasis. To investigate the relationship between ECM genes and poor prognosis in breast cancer, expression quantitative trait locus analysis of polyoma middle-T transgene-induced mammary tumor was performed. ECM gene expression was found to be consistently associated with Rrp1b expression. In vitro expression of Rrp1b significantly altered ECM gene expression, tumor growth, and dissemination in metastasis assays. Furthermore, a gene signature induced by ectopic expression of Rrp1b in tumor cells predicted survival in a human breast cancer gene expression dataset. Finally, constitutional polymorphism within RRP1B was found to be significantly associated with tumor progression in two independent breast cancer cohorts. These data suggest that RRP1B may be a novel susceptibility gene for breast cancer progression and metastasis.     

Investigating the antiproliferative activity of quinoline-5,8-diones and styrylquinolinecarboxylic acids on tumor cell lines

The structure-activity relationships of new quinoline based compounds were investigated. Quinoline-5,8-dione and styrylquinoline scaffolds were used for the design of potentially active compounds. The novel analogues had comparable antiproliferative activity to cisplatin when evaluated in a bioassay against the P388 leukemia cell line. However, these compounds appeared far less efficient against SK-N-MC neuroepithelioma cells. Analogues without the 5,8-dione structure but containing the 8-carboxylic acid group were also found to induce antiproliferative activity. Hydrophobicity as measured by HPLC did not correlate with antiproliferative activity.     

The Tubular Sheaths Encasing Methanosaeta thermophila Filaments Are Functional Amyloids

Archaea are renowned for their ability to thrive in extreme environments, although they can be found in virtually all habitats. Their adaptive success is linked to their unique cell envelopes that are extremely resistant to chemical and thermal denaturation and that resist proteolysis by common proteases. Here we employ amyloid-specific conformation antibodies and biophysical techniques to show that the extracellular cell wall sheaths encasing the methanogenic archaea Methanosaeta thermophila PT are functional amyloids. Depolymerization of sheaths and subsequent MS/MS analyses revealed that the sheaths are composed of a single major sheath protein (MspA). The amyloidogenic nature of MspA was confirmed by in vitro amyloid formation of recombinant MspA under a wide range of environmental conditions. This is the first report of a functional amyloid from the archaeal domain of life. The amyloid nature explains the extreme resistance of the sheath, the elastic properties that allow diffusible substrates to penetrate through expandable hoop boundaries, and how the sheaths are able to split and elongate outside the cell. The archaeal sheath amyloids do not share homology with any of the currently known functional amyloids and clearly represent a new function of the amyloid protein fold.     

Novel small molecule inhibitors of 3-phosphoinositide-dependent kinase-1

The phosphoinositide 3-kinase/3-phosphoinositide-dependent kinase 1 (PDK1)/Akt signaling pathway plays a key role in cancer cell growth, survival, and tumor angiogenesis and represents a promising target for anticancer drugs. Here, we describe three potent PDK1 inhibitors, BX-795, BX-912, and BX-320 (IC(50) = 11-30 nm) and their initial biological characterization. The inhibitors blocked PDK1/Akt signaling in tumor cells and inhibited the anchorage-dependent growth of a variety of tumor cell lines in culture or induced apoptosis. A number of cancer cell lines with elevated Akt activity were >30-fold more sensitive to growth inhibition by PDK1 inhibitors in soft agar than on tissue culture plastic, consistent with the cell survival function of the PDK1/Akt signaling pathway, which is particularly important for unattached cells. BX-320 inhibited the growth of LOX melanoma tumors in the lungs of nude mice after injection of tumor cells into the tail vein. The effect of BX-320 on cancer cell growth in vitro and in vivo indicates that PDK1 inhibitors may have clinical utility as anticancer agents.     

Protein 4.2 Binds to the Carboxyl-terminal EF-hands of Erythroid ��-Spectrin in a Calcium- and Calmodulin-dependent Manner

Spectrin and protein 4.1 cross-link F-actin protofilaments into a network called the membrane skeleton. Actin and 4.1 bind to one end of β-spectrin. The adjacent end of α-spectrin, called the EF-domain, is calmodulin-like, with calcium-dependent and calcium-independent EF-hands. It has no known function. However, the sph^1J/sph^1J mouse has very fragile red cells and lacks the last 13 amino acids in the EF-domain, suggesting the domain is critical for skeletal integrity. Using pulldown binding assays, we find the α-spectrin EF-domain either alone or incorporated into a mini-spectrin binds native and recombinant protein 4.2 at a previously identified region of 4.2 (G₃ peptide). Native 4.2 binds with an affinity comparable with other membrane skeletal interactions (K_d = 0.30 μm). EF-domains bearing the sph^1J mutation are inactive. Binding of protein 4.2 to band 3 (K_d = 0.45 μm) does not interfere with the spectrin-4.2 interaction. Spectrin-4.2 binding is amplified by micromolar concentrations of Ca²⁺ (but not Mg²⁺) by three to five times. Calmodulin also binds to the EF-domain (K_d = 17 μm), and Ca²⁺-calmodulin blocks Ca²⁺-dependent binding of protein 4.2 but not Ca²⁺-independent binding. The data suggest that protein 4.2 is located near protein 4.1 at the spectrin-actin junctions. Because proteins 4.1 and 4.2 also bind to band 3, the erythrocyte anion channel, we suggest that one or both of these proteins cause a portion of band 3 to localize near the spectrin-actin junctions and provide another point of attachment between the membrane skeleton and the lipid bilayer.