In vivo random mutagenesis of streptomycetes using mariner-based transposon Himar1

We report here the in vivo expression of the synthetic transposase gene himar1(a) in Streptomyces coelicolor M145 and Streptomyces albus. Using the synthetic himar1(a) gene adapted for Streptomyces codon usage, we showed random insertion of the transposon into the streptomycetes genome. The insertion frequency for the Himar1-derived minitransposons is nearly 100 % of transformed Streptomyces cells, and insertions are stably inherited in the absence of an antibiotic selection. The minitransposons contain different antibiotic resistance selection markers (apramycin, hygromycin, and spectinomycin), site-specific recombinase target sites (rox and/or loxP), I-SceI meganuclease target sites, and an R6Kγ origin of replication for transposon rescue. We identified transposon insertion loci by random sequencing of more than 100 rescue plasmids. The majority of insertions were mapped to putative open-reading frames on the S. coelicolor M145 and S. albus chromosomes. These insertions included several new regulatory genes affecting S. coelicolor M145 growth and actinorhodin biosynthesis.     

Mutations in BICD2, which Encodes a Golgin and Important Motor Adaptor,Cause Congenital Autosomal-Dominant Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is a heterogeneous group of neuromuscular disorders caused by degeneration of lower motor neurons. Although functional loss of SMN1 is associated with autosomal-recessive childhood SMA, the genetic cause for most families affected by dominantly inherited SMA is unknown. Here, we identified pathogenic variants in bicaudal D homolog 2 (Drosophila) (BICD2) in three families afflicted with autosomal-dominant SMA. Affected individuals displayed congenital slowly progressive muscle weakness mainly of the lower limbs and congenital contractures. In a large Dutch family, linkage analysis identified a 9q22.3 locus in which exome sequencing uncovered c.320C>T (p.Ser107Leu) in BICD2. Sequencing of 23 additional families affected by dominant SMA led to the identification of pathogenic variants in one family from Canada (c.2108C>T [p.Thr703Met]) and one from the Netherlands (c.563A>C [p.Asn188Thr]). BICD2 is a golgin and motor-adaptor protein involved in Golgi dynamics and vesicular and mRNA transport. Transient transfection of HeLa cells with all three mutant BICD2 cDNAs caused massive Golgi fragmentation. This observation was even more prominent in primary fibroblasts from an individual harboring c.2108C>T (p.Thr703Met) (affecting the C-terminal coiled-coil domain) and slightly less evident in individuals with c.563A>C (p.Asn188Thr) (affecting the N-terminal coiled-coil domain). Furthermore, BICD2 levels were reduced in affected individuals and trapped within the fragmented Golgi. Previous studies have shown that Drosophila mutant BicD causes reduced larvae locomotion by impaired clathrin-mediated synaptic endocytosis in neuromuscular junctions. These data emphasize the relevance of BICD2 in synaptic-vesicle recycling and support the conclusion that BICD2 mutations cause congenital slowly progressive dominant SMA.     

S-adenosyl-L-homocysteine hydrolase and methylation disorders: Yeast as a model system

S-adenosyl-L-methionine (AdoMet)-dependent methylation is central to the regulation of many biological processes: more than 50 AdoMet-dependent methyltransferases methylate a broad spectrum of cellular compounds including nucleic acids, proteins and lipids. Common to all AdoMet-dependent methyltransferase reactions is the release of the strong product inhibitor S-adenosyl-L-homocysteine (AdoHcy), as a by-product of the reaction. S-adenosyl-L-homocysteine hydrolase is the only eukaryotic enzyme capable of reversible AdoHcy hydrolysis to adenosine and homocysteine and, thus, relief from AdoHcy inhibition. Impaired S-adenosyl-L-homocysteine hydrolase activity in humans results in AdoHcy accumulation and severe pathological consequences. Hyperhomocysteinemia, which is characterized by elevated levels of homocysteine in blood, also exhibits a similar phenotype of AdoHcy accumulation due to the reversal of the direction of the S-adenosyl-L-homocysteine hydrolase reaction. Inhibition of S-adenosyl-L-homocysteine hydrolase is also linked to antiviral effects. In this review the advantages of yeast as an experimental system to understand pathologies associated with AdoHcy accumulation will be discussed.     

Synthesis and Antioxidant Activity of Carane and Pinane Based Sulfenimines and Sulfinimines

The synthesis of sulfenimines and sulfinimines has been carried out with 10‐hydroxyisocamphylthiol. The configuration of the compounds has been deduced by methods of NMR, DFT calculations and X‐ray diffraction analysis. The cytotoxic, antioxidant and membrane‐protective activity of the synthesized compounds as well as of the previously obtained sulfenimines and sulfinimines based on 4‐caranethiol have been determined.     

Functioning of plant-bacterial associations under osmotic stress in vitro

World Journal of Microbiology and Biotechnology - Derived from RNA, 5?-ribonucleotides, especially Inosine-5?-monophosphate (IMP) and guanosine-5?-monophosphate (GMP), can enhance...     

The stereochemistry of benzo[a]pyrene-2'-deoxyguanosine adducts affects DNA methylation by SssI and HhaI DNA methyltransferases

The biologically most significant genotoxic metabolite of the environmental pollutant benzo[a]pyrene (B[a]P), (+)-7R,8S-diol 9S,10R-epoxide, reacts chemically with guanine in DNA, resulting in the predominant formation of (+)-trans-B[a]P-N(2)-dG and, to a lesser extent, (+)-cis-B[a]P-N(2)-dG adducts. Here, we compare the effects of the adduct stereochemistry and conformation on the methylation of cytosine catalyzed by two purified prokaryotic DNA methyltransferases (MTases), SssI and HhaI, with the lesions positioned within or adjacent to their CG and GCGC recognition sites, respectively. The fluorescence properties of the pyrenyl residues of the (+)-cis-B[a]P-N(2)-dG and (+)-trans-B[a]P-N(2)-dG adducts in complexes with MTases are enhanced, but to different extents, indicating that aromatic B[a]P residues are positioned in different microenvironments in the DNA-protein complexes. We have previously shown that the (+)-trans-isomeric adduct inhibits both the binding and methylating efficiencies (k(cat)) of both MTases [Subach OM, Baskunov VB, Darii MV, Maltseva DV, Alexandrov DA, Kirsanova OV, Kolbanovskiy A, Kolbanovskiy M, Johnson F, Bonala R, et al. (2006) Biochemistry45, 6142-6159]. Here we show that the stereoisomeric (+)-cis-B[a]P-N(2)-dG lesion has only a minimal effect on the binding of these MTases and on k(cat). The minor-groove (+)-trans adduct interferes with the formation of the normal DNA minor-groove contacts with the catalytic loop of the MTases. However, the intercalated base-displaced (+)-cis adduct does not interfere with the minor-groove DNA-catalytic loop contacts, allowing near-normal binding of the MTases and undiminished k(cat) values.     

Phytohormone Priming: Regulator for Heavy Metal Stress in Plants

Phytohormones act as chemical messengers and, under a complex regulation, allow plants to sustain biotic and abiotic stresses. Thus, phytohormones are known for their regulatory role in plant growth and development. Heavy metals (HMs) play an important role in metabolism and have roles in plant growth and development as micronutrients. However, at a level above threshold, these HMs act as contaminants and pose a worldwide environmental threat. Thus, finding eco-friendly and economical deliverables to tackle this problem is a priority. In addition to physicochemical methods, exogenous application of phytohormones, i.e., auxins, cytokinins, and gibberellins, can positively influence the regulation of the ascorbate–glutathione cycle, transpiration rate, cell division, and the activities of nitrogen metabolism and assimilation, which improve plant growth activity. Brassinosteroids, ethylene and salicylic acid have been reported to enhance the level of the anti-oxidant system, decrease levels of ROS, lipid peroxidation and improve photosynthesis in plants, when applied exogenously under a HM effect. There is a crosstalk between phytohormones which is activated upon exogenous application. Research suggests that plants are primed by phytohormones for stress tolerance. Chemical priming has provided good results in plant physiology and stress adaptation, and phytohormone priming is underway. We have reviewed promising phytohormones, which can potentially confer enhanced tolerance when used exogenously. Exogenous application of phytohormones may increase plant performance under HM stress and can be used for agro-ecological benefits under environmental conditions with high HMs level.

     

Mechanical stretch and progesterone differentially regulate activator protein-1 transcription factors in primary rat myometrial smooth muscle cells

During pregnancy, stretch of the uterus, imposed by the growing fetus, is an important signal for the induction of genes involved in the onset of labor. In this study, the expression of activator protein-1 (AP-1) family mRNAs in response to in vitro stretch was investigated in myometrial cells. Rat primary myometrial smooth muscle cells were plated onto collagen I-coated Flex I culture plates and subjected to 25% static stretch on day 4 of culture. Static stretch induced an increase in the expression of c-fos, fosB, fra-1, c-jun, and junB. The expression of both c-fos and junB was maximally induced at 30 min by static stretch. The peak induction for fosB and c-jun occurred at 1 h, whereas the peak of fra-1 induction occurred between 1 and 2 h after application of stretch. Treatment of myometrial cells with progesterone (100 nM, 400 nM, 1 microM) for 1 or 6 h before the application of static stretch did not affect the magnitude of the c-fos response. However, 24 h of progesterone exposure reduced the magnitude of c-fos and fosB stretch induction at both the 400 nM and 1 microM doses. These data indicate that several members of the AP-1 family are stretch-responsive genes in myometrial smooth muscle cells. This response can be attenuated by pretreatment with progesterone; however, the requirement for longer pretreatment times suggests that the inhibitory actions of progesterone do not occur through a direct action of the progesterone receptor within the promoter regions of AP-1 genes.     

Flagellar radial spokes contain a Ca2+-stimulated nucleoside diphosphate kinase

The radial spokes are required for Ca(2+)-initiated intraflagellar signaling, resulting in modulation of inner and outer arm dynein activity. However, the mechanochemical properties of this signaling pathway remain unknown. Here, we describe a novel nucleoside diphosphate kinase (NDK) from the Chlamydomonas flagellum. This protein (termed p61 or RSP23) consists of an N-terminal catalytic NDK domain followed by a repetitive region that includes three IQ motifs and a highly acidic C-terminal segment. We find that p61 is missing in axonemes derived from the mutants pf14 (lacks radial spokes) and pf24 (lacks the spoke head and several stalk components) but not in those from pf17 (lacking only the spoke head). The p61 protein can be extracted from oda1 (lacks outer dynein arms) and pf17 axonemes with 0.5 M KI, and copurifies with radial spokes in sucrose density gradients. Furthermore, p61 contains two classes of calmodulin binding site: IQ1 interacts with calmodulin-Sepharose beads in a Ca(2+)-independent manner, whereas IQ2 and IQ3 show Ca(2+)-sensitive associations. Wild-type axonemes exhibit two distinct NDKase activities, at least one of which is stimulated by Ca(2+). This Ca(2+)-responsive enzyme, which accounts for approximately 45% of total axonemal NDKase, is missing from pf14 axonemes. We found that purified radial spokes also exhibit NDKase activity. Thus, we conclude that p61 is an integral component of the radial spoke stalk that binds calmodulin and exhibits Ca(2+)-controlled NDKase activity. These observations suggest that nucleotides other than ATP may play an important role in the signal transduction pathway that underlies the regulatory mechanism defined by the radial spokes.     

Occurrence and activity of Archaea in aerated activated sludge wastewater treatment plants

The occurrence, distribution and activity of archaeal populations within two aerated, activated sludge wastewater treatment systems, one treating domestic waste and the second treating mixed domestic and industrial wastewater, were investigated by denaturing gradient gel electrophoresis (DGGE) analysis of polymerase chain reaction (PCR)-amplified ribosomal RNA gene fragments and process measurements. In the plant receiving mixed industrial and domestic waste the archaeal populations found in the mixed liquor were very similar to those in the influent sewage, though a small number of DGGE bands specific to the mixed liquor were identified. In contrast, the activated sludge treating principally domestic waste harboured distinct archaeal populations associated with the mixed liquor that were not prevalent in the influent sewage. We deduce that the Archaea in the plant treating mixed wastewater were derived principally from the influent, whereas those in the plant treating solely domestic waste were actively growing in the treatment plant. Archaeal 16S rRNA gene sequences related to the Methanosarcinales, Methanomicrobiales and the Methanobacteriales were detected. Methanogenesis was measured in activated sludge samples incubated under oxic and anoxic conditions, demonstrating that the methanogens present in both activated sludge plants were active only in anoxic incubations. The relatively low rates of methanogenesis measured indicated that, although active, the methanogens play a minor role in carbon turnover in activated sludge.