Synthesis of Non-Natural Pyrimidine Nucleosides

Abstract

Two new non-natural nucleosides bearing an amide (8) or an amidine (9) function have been synthesized. Their properties and the geometry of the exocyclic double bond have been studied.     

Ancylobacter lacus sp. nov. and Ancylobacter plantiphilus sp. nov., Novel Aerobic Facultative Methylotrophic Bacteria

Microbiology - Novel aerobic facultatively methylotrophic bacteria were isolated from the water of a freshwater lake (strain F30LT), soil sample of rhizosphere of white clover Trifolium repens L....     

Reclassification of Methylarcula marina Doronina et al. 2000 as Paracoccus methylarcula nom. nov.

Microbiology - Phylogenetic analysis of the 16S rRNA gene sequences showed that the type species of the genus Methylarcula, Methylarculamarina h1T was closely related to Paracoccus saliphilus DSM...     

Halophilic and halotolerant aerobic methylobacteria from the technogenic Solikamsk biotopes

Seven strains of moderately halophilic and halotolerant aerobic methylobacteria from the technogenic Solikamsk biotopes (Perm krai, Russia) were isolated in pure cultures and characterized. The isolates were represented by gram-negative and gram-positive (strain 2395B) cells. All the cells were shown to multiply by binary fission without formation of spores or prosthecae. All isolates except strain 2395B were able to oxidize methanol by a classical methanol dehydrogenase. The ribulose monophosphate (RMP) (strain LS), serine (strains S12, S3, 2395A), or ribulose bisphosphate (strains SK15 and S3270) pathways of C₁-assimilation were used. In strain 2395B, the key enzymes of the RMP and serine metabolic pathways were determined. Using polyphasic taxonomy, three strains were identified as representatives of the known species: Arthrobacter protophormiae 2395B, Methylophaga thalassica LS, and Ancylobacter rudongensis S3270. Three more strains were identified as members of new species: Methylopila oligotropha sp. nov. (strain 2395AT; VKM B-2788^T = CCUG 63805^T), Ancylobacter defluvii sp. nov. (strain SK15^T; VKM B-2789^T = CCUG 63806^T), and Paracoccus communis sp. nov. (strain S3^T; VKM B-2787^T = CCUG 63804^T). According to the results of 16S rRNA gene sequencing, the obligately methylotrophic strain S12 had less than 94% similarity with the known genera of the Proteobacteria and was probably a representative of a novel genus.     

Inhibition of TRAIL-induced apoptosis and forced internalization of TRAIL receptor 1 by adenovirus proteins

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptosis through two receptors, TRAIL-R1 (also known as death receptor 4) and TRAIL-R2 (also known as death receptor 5), that are members of the TNF receptor superfamily of death domain-containing receptors. We show that human adenovirus type 5 encodes three proteins, named RID (previously named E3-10.4K/14.5K), E3-14.7K, and E1B-19K, that independently inhibit TRAIL-induced apoptosis of infected human cells. This conclusion was derived from studies using wild-type adenovirus, adenovirus replication-competent mutants that lack one or more of the RID, E3-14.7K, and E1B-19K genes, and adenovirus E1-minus replication-defective vectors that express all E3 genes, RID plus E3-14.7K only, RID only, or E3-14.7K only. RID inhibits TRAIL-induced apoptosis when cells are sensitized to TRAIL either by adenovirus infection or treatment with cycloheximide. RID induces the internalization of TRAIL-R1 from the cell surface, as shown by flow cytometry and indirect immunofluorescence for TRAIL-R1. TRAIL-R1 was internalized in distinct vesicles which are very likely to be endosomes and lysosomes. TRAIL-R1 is degraded, as indicated by the disappearance of the TRAIL-R1 immunofluorescence signal. Degradation was inhibited by bafilomycin A1, a drug that prevents acidification of vesicles and the sorting of receptors from late endosomes to lysosomes, implying that degradation occurs in lysosomes. RID was also shown previously to internalize and degrade another death domain receptor, Fas, and to prevent apoptosis through Fas and the TNF receptor. RID was shown previously to force the internalization and degradation of the epidermal growth factor receptor. E1B-19K was shown previously to block apoptosis through Fas, and both E1B-19K and E3-14.7K were found to prevent apoptosis through the TNF receptor. These findings suggest that the receptors for TRAIL, Fas ligand, and TNF play a role in limiting virus infections. The ability of adenovirus to inhibit killing through these receptors may prolong acute and persistent infections.     

Novel Aerobic Methylotrophic Isolates from the Soda Lakes of the Southern Transbaikal Region

Twenty-one bacterial associations isolated from the soda lakes of the southern Transbaikal region were found to be able to actively grow at pH 9–10 on methanol as the source of carbon and energy. Two alkalitolerant facultatively methylotrophic strains, Bur 3 and Bur 5, were obtained in pure cultures. Both strains represent gram-negative, nonmotile, bean-shaped, encapsulated cells that reproduce by binary fission. The strains are able to grow at temperatures ranging from 6 to 42°C, with an optimum growth temperature of 25–29°C (strain Bur 3) and 35–37°C (strain Bur 5) and at pH between 6.5 and 9.5, with an optimum pH value of 8.0–8.5. At pH 9.0, strain Bur 3 exhibits an increased content of phosphatidylglycerol and a decreased content of phosphatidylethanolamine. Strains Bur 3 and Bur 5 are similar in the G+C content of their DNAs (66.2 and 65.5 mol %, respectively) and in the type of the dominant ubiquinone (Q ₁₀). Unlike Bur 5, strain Bur 3 is able to grow autotrophically in an atmosphere of CO₂+ O₂+ H₂. The strains oxidize, by the respective dehydrogenases, methanol to CO₂, which is assimilated by the ribulose bisphosphate pathway. Ammonium ions are assimilated in the glutamate cycle and by the reductive amination of -ketoglutarate. The strains are highly homologous to each other (92%) and are much less homologous (at a level of 28–35%) to representatives of the genus Ancylobacter, A. aquaticusATCC 25396^Tand A. vacuolatumDSM 1277. Based on the results obtained, both strains are assigned to a new species, Ancylobacter natronumsp. nov.     

The proteolytic control of restriction activity in Escherichia coli K-12

The endonuclease activity of EcoKI is regulated by the ClpXP-dependent degradation of the subunit that is essential for restriction, but not modification. We monitored proteolysis in mutants blocked at different steps in the restriction pathway. Mutations that prevent DNA translocation render EcoKI refractory to proteolysis, whereas those that permit DNA translocation, but block endonuclease activity, do not. Although proteolysis alleviates restriction in a mutant that lacks modification activity, some restriction activity remains; our evidence indicates residual EcoKI associated with the membrane fraction. ClpXP protects the bacterial chromosome, but little effect was detected on unmodified foreign DNA within the cytoplasm of a restriction-proficient cell. The molecular basis for the distinction between unmodified resident and foreign DNA remains to be determined.     

Efficient synthesis of oligonucleotide-peptide conjugates on large scale

The conjugation of oligonucleotide phosphorothioates with antennapedia peptide was studied in detail to allow efficient preparation of the conjugates on up to 15 mumol scale. Under optimized conditions, the use of oligonucleotides and the peptide in an equimolecular ratio gave the desired conjugates in more than 60% isolated yield.     

A novel plant-associated thermotolerant alkalophilic methylotroph of the genus Paracoccus

Strain GB isolated from the maize rhizosphere is a gram-negative, aerobic, non-spore-forming, nonpigmented, nonmotile, chemolithotrophic, facultatively methylotrophic bacterium. Cells are cocci or short rods. The strain does not require vitamins. Optimum growth in a medium with methanol occurs at 38-42 degrees C at pH 8.0-9.2. The doubling time is 12 h. In addition to methanol, the bacterium can grow on methylamine, dimethylformamide, acetone, thiosulfate + NaHCO3, and in an atmosphere of H2 + CO2 + O2. Methanol and methylamine are oxidized by the respective dehydrogenases to CO2 via formaldehyde and formate, respectively. The CO2 produced is assimilated via the ribulose bisphosphate pathway. Fatty acids are dominated by cyclopropanoic (58-61%), palmitic (24-26%), and octadecanoic (8-9%) acids. The main phospholipids are phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylcholine. The major ubiquinone is Q10. The bacterial genome contains genes controlling the synthesis and secretion of cytokinins. The culture liquid exhibits cytokinin activity. The G + C content of DNA is 62.5 mol %, as determined from the DNA thermal denaturation temperature (Tm). Strain GB shows a moderate degree of DNA-DNA homology (< 40%) with the type representatives of the genus Paracoccus. Based on the data obtained, the bacterium was classified as a new species of this genus, named P. kondratievae.     

Orchestrating ribosomal activity from inside: effects of the nascent chain on the peptidyltransferase centre

Ribosomal progression through the open reading frames within mRNAs is frequently considered as uneventful when compared with the highly regulated initiation step. However, both RNA and nascent peptide can interact with the ribosome to influence how translation proceeds and can modify gene expression in several ways. 2A peptides are a class of sequences that, as nascent chains, pause ribosomes and drive a translation-termination reaction on a sense (proline) codon, followed by continued downstream translation. In the present paper, what is known about the 2A reaction is discussed, and 2A is compared with other sequences that, as nascent peptides, pause or stall translation.