Variation in organisation and copy number of ribosomal RNA genes inPetunia hybrida somaclones

The copy number of genes encoding 5S ribosomal RNA has been found to be constant in Petunia hybrida plants regenerated from protoplast and leaf disc-derived callus cultures. However, in one somaclone a heritable change in the length of the major 5S rDNA repeat has arisen. Despite the constant copy number of 5S rRNA genes, that of the 18S–25S rRNA genes is found to very by at least ten-fold. The relevance of these findings to ribosomal RNA gene variability and to somaclonal variation is discussed.     

Mutant forms of Escherichia coli protein L25 unable to bind to 5S rRNA are incorporated efficiently into the ribosome in vivo

5S rRNA-binding ribosomal proteins of the L25 family are an evolutional acquisition of bacteria. Earlier we showed that (i) single replacements in the RNA-binding module of the protein of this family result in destabilization or complete impossibility to form a complex with 5S rRNA in vitro; (ii) ΔL25 ribosomes of Escherichia coli are less efficient in protein synthesis in vivo than the control ribosomes. In the present work, the efficiency of incorporation of the E. coli protein L25 with mutations in the 5S rRNA-binding region into the ribosome in vivo was studied. It was found that the mutations in L25 that abolish its ability to form the complex with free 5S rRNA do not prevent its correct and efficient incorporation into the ribosome. This is supported by the fact that even the presence of a very weakly retained mutant form of the protein in the ribosome has a positive effect on the activity of the translational machinery in vivo. All this suggests the existence of an alternative incorporation pathway for this protein into the ribosome, excluding the preliminary formation of the complex with 5S rRNA. At the same time, the stable L25-5S rRNA contact is important for the retention of the protein within the ribosome, and the conservative amino acid residues of the RNA-binding module play a key role in this.     

Size class homogeneity of repeat lengths and evolutionary divergence of ribosomal RNA genes in fishes as studied by restriction fragment length analysis

Fish ribosomal RNA genes (rDNA) have been compared by restriction endonuclease digestion followed by Southern hybridization using rRNA or cloned rRNA genes as labelled probes. In several species belonging to the orders Cypriniformes and Perciformes, the simple restriction patterns revealed a high degree of size class homogeneity among the rDNA repeats and similar restriction map within a species. Different species have different restriction patterns and fragment lengths arising mostly out of different length of the nontranscribed spacer. Polymorphic restriction sites are present in some species. The species-specific differences in fragment lengths produced in rDNA by some restriction enzymes can thus be used to study interspecific fish hybrids.     

The evolutionary pattern of aromatic amino acid biosynthesis and the emerging phylogeny of pseudomonad bacteria

Pseudomonad bacterial are a phylogenetically diverse assemblage of species named within contemporary genera that includePseudomonas, Xanthomonas andAlcaligenes. Thus far, five distinct rRNA homology groups (Groups I through V) have been established by oligonucleotide cataloging and by rRNA/DNA hybridization. A pattern of enzymic features of aromatic amino acid biosynthesis (enzymological patterning) is conserved at the level of rRNA homology, five distinct and unambiguous patterns therefore existing in correspondence with the rRNA homology groups. We sorted 87 pseudomonad strains into Groups (and Subgroups) by aromatic pathway patterning. The reliability of this methodology was tested in a blind study using coded cultures of diverse pseudomonad organisms provided by American Type Culture Collection. Fourteen of 14 correct assignments were made at the Group level (the level of rRNA homology), and 12 of 14 correct assignments were made at the finer-tuned Subgroup levels. Many strains of unknown rRNA-homology affiliation had been placed into tentative rRNA groupings based upon enzymological patterning. Positive confirmation of such strains as members of the predicted rRNA homology groups was demonstrated by DNA/rRNA hybridization in nearly every case. It seems clear that the combination of these molecular approaches will make it feasible to deduce the evolution of biochemical-pathway construction and regulation in parallel with the emerging phylogenies of microbes housing these pathways.     

The Neuroprotective Effects of α-Iso-cubebene on Dopaminergic Cell Death: Involvement of CREB/Nrf2 Signaling

As a part of ongoing studies to elucidate pharmacologically active components of Schisandra chinensis, we isolated and studied α-iso-cubebene. The neuroprotective mechanisms of α-iso-cubebene in human neuroblastoma SH-SY5Y cells were investigated. α-Iso-cubebene significantly inhibited cytotoxicity and apoptosis due to 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in dopaminergic SH-SY5Y cells. Pretreatment of cells with α-iso-cubebene reduced intracellular accumulation of ROS and calcium in response to 6-OHDA. The neuroprotective effects of α-iso-cubebene were found to result from protecting the mitochondrial membrane potential. Notably, α-iso-cubebene inhibited the release of apoptosis-inducing factor from the mitochondria into the cytosol and nucleus after 6-OHDA treatment. α-Iso-cubebene also induced the activation of PKA/PKB/CREB/Nrf2 and suppressed 6-OHDA-induced neurotoxicity. α-Iso-cubebene was found to induce phosphorylation of PKA and PKB and activate Nrf2 and CREB signaling pathways in a dose-dependent manner. Additionally, α-iso-cubebene stimulated the expression of the antioxidant response genes NQO1 and HO-1. Finally, α-iso-cubebene-mediated neuroprotective effects were found to be reversible after transfection with CREB and Nrf2 small interfering RNAs.     

The neurobiological link between OCD and ADHD

Obsessive compulsive disorder (OCD) and attention deficit hyperactivity disorder (ADHD) are two of the most common neuropsychiatric diseases in paediatric populations. The high comorbidity of ADHD and OCD with each other, especially of ADHD in paediatric OCD, is well described. OCD and ADHD often follow a chronic course with persistent rates of at least 40–50 %. Family studies showed high heritability in ADHD and OCD, and some genetic findings showed similar variants for both disorders of the same pathogenetic mechanisms, whereas other genetic findings may differentiate between ADHD and OCD. Neuropsychological and neuroimaging studies suggest that partly similar executive functions are affected in both disorders. The deficits in the corresponding brain networks may be responsible for the perseverative, compulsive symptoms in OCD but also for the disinhibited and impulsive symptoms characterizing ADHD. This article reviews the current literature of neuroimaging, neurochemical circuitry, neuropsychological and genetic findings considering similarities as well as differences between OCD and ADHD.     

Crystal structure of tRNA m1A58 methyltransferase TrmI from Aquifex aeolicus in complex with S-adenosyl-l-methionine

The N ¹-methyladenosine residue at position 58 of tRNA is found in the three domains of life, and contributes to the stability of the three-dimensional L-shaped tRNA structure. In thermophilic bacteria, this modification is important for thermal adaptation, and is catalyzed by the tRNA m¹A58 methyltransferase TrmI, using S-adenosyl-l-methionine (AdoMet) as the methyl donor. We present the 2.2 Å crystal structure of TrmI from the extremely thermophilic bacterium Aquifex aeolicus, in complex with AdoMet. There are four molecules per asymmetric unit, and they form a tetramer. Based on a comparison of the AdoMet binding mode of A. aeolicus TrmI to those of the Thermus thermophilus and Pyrococcus abyssi TrmIs, we discuss their similarities and differences. Although the binding modes to the N6 amino group of the adenine moiety of AdoMet are similar, using the side chains of acidic residues as well as hydrogen bonds, the positions of the amino acid residues involved in binding are diverse among the TrmIs from A. aeolicus, T. thermophilus, and P. abyssi.     

Detection of polyketide synthase and nonribosomal peptide synthetase biosynthetic genes from antimicrobial coral-associated actinomycetes

The diversity and properties of actinobacteria, predominant residents in coral holobionts, have been rarely documented. In this study, we aimed to explore the species diversity, antimicrobial activities and biosynthetic potential of culturable actinomycetes within the tissues of the scleractinian corals Porites lutea, Galaxea fascicularis and Acropora millepora from the South China Sea. A total of 70 strains representing 13 families and 15 genera of actinobacteria were isolated. The antimicrobial activity and biosynthetic potential of fifteen representative filamentous actinomycetes were estimated. Crude fermentation extracts of 6 strains exhibited comparable or greater activities against Vibrio alginolyticus than ciprofloxacin. Seven of the 15 actinomycetes strains possess type I polyketide synthases (PKS-I) and/or nonribosomal peptide synthetases (NRPS) genes. Nine tested strains possess type II polyketide synthases (PKS-II). Phylogenetic analysis based on 16S rRNA gene sequences indicated that these PKS and NRPS gene screening positive strains belong to genera Nocardiopsis, Pseudonocardia, Streptomyces, Micromonospora, Amycolatopsis and Prauserella. One PKS-I and four NRPS fragments showed <70 % similarity to their closest relatives, which suggested the novelty of these genes. This study helps uncover the genetic capacity of stony coral-associated actinomycetes to produce bioactive molecules.     

Urokinase-type plasminogen activator receptor regulates apoptotic sensitivity of colon cancer HCT116 cell line to TRAIL via JNK-p53 pathway

The urokinase-type plasminogen activator receptor (uPAR) serves not only as an anchor for urokinase-type plasminogen activator but also participates in intracellular signal transduction events. In this study, we investigated whether uPAR could modulate TRAIL-induced apoptosis in human colon cancer cells HCT116. Using an antisense strategy, we established a stable HCT116 cell line with down-regulated uPAR. The sensitivity to TRAIL-induced apoptosis was evaluated by FACS analysis. Our results show that the inhibition of uPAR could sensitize HCT116 to TRAIL-induced apoptosis. uPAR inhibition changed the expression of mitochondrial apoptotic pathway proteins, including Bcl-2, Bax, Bid and p53, in a pro-apoptotic manner. We also found that the inhibition of uPAR down-regulated the phosphorylation of FAK, ERK and JNK. The inhibition of p53 by RNA interference rescued cells from enhanced apoptosis, thus indicating that p53 is critical for enhancing TRAIL-induced apoptosis. Furthermore, JNK, but not ERK, inhibition involved in the up-regulation of p53. JNK negatively regulated p53 protein level. Overall, our results show that uPAR inhibition can sensitize colon cancer cells HCT116 to TRAIL-induced apoptosis via active p53 and mitochondrial apoptotic pathways that JNK inhibition is involved.     

Burkholderia sp. Induces Functional Nodules on the South African Invasive Legume Dipogon lignosus (Phaseoleae) in New Zealand Soils

The South African invasive legume Dipogon lignosus (Phaseoleae) produces nodules with both determinate and indeterminate characteristics in New Zealand (NZ) soils. Ten bacterial isolates produced functional nodules on D. lignosus. The 16S ribosomal RNA (rRNA) gene sequences identified one isolate as Bradyrhizobium sp., one isolate as Rhizobium sp. and eight isolates as Burkholderia sp. The Bradyrhizobium sp. and Rhizobium sp. 16S rRNA sequences were identical to those of strains previously isolated from crop plants and may have originated from inocula used on crops. Both 16S rRNA and DNA recombinase A (recA) gene sequences placed the eight Burkholderia isolates separate from previously described Burkholderia rhizobial species. However, the isolates showed a very close relationship to Burkholderia rhizobial strains isolated from South African plants with respect to their nitrogenase iron protein (nifH), N-acyltransferase nodulation protein A (nodA) and N-acetylglucosaminyl transferase nodulation protein C (nodC) gene sequences. Gene sequences and enterobacterial repetitive intergenic consensus (ERIC) PCR and repetitive element palindromic PCR (rep-PCR) banding patterns indicated that the eight Burkholderia isolates separated into five clones of one strain and three of another. One strain was tested and shown to produce functional nodules on a range of South African plants previously reported to be nodulated by Burkholderia tuberum STM678^T which was isolated from the Cape Region. Thus, evidence is strong that the Burkholderia strains isolated here originated in South Africa and were somehow transported with the plants from their native habitat to NZ. It is possible that the strains are of a new species capable of nodulating legumes.     

Pyrosequencing-based analysis of fecal microbial communities in three purebred pig lines

This study examined the fecal bacterial diversity of 15-weekold pigs from three purebred lines: Duroc, Landrace, and Yorkshire. Taxon-dependent and -independent analyses were performed to evaluate differences in the fecal bacterial communities and to identify bacterial genera that can be used to discriminate breeds, following high-throughput pyrosequencing of 16S rRNA genes. Among the breeds evaluated, Landrace had the most diverse bacterial community composition. Prevotella, Blautia, Oscillibacter, and Clostridium were detected in all samples regardless of breed. On the other hand, Catenibacterium, Blautia, Dialister, and Sphaerochaeta were differentially detected among breeds, as demonstrated by the canonical loading plot. The discriminant analysis of principal components plot also showed clear separation of the three purebred pig lines, with a certain degree of similarity between Landrace and Yorkshire pigs and a distinct separation between Duroc pigs and the other two breeds. Other factors not related to breed, such as season or time of sampling and pen effects, may contribute to shaping the gut microbiota of pigs.     

Modeling progressive non-alcoholic fatty liver disease in the laboratory mouse

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in the world and its prevalence is rising. In the absence of disease progression, fatty liver poses minimal risk of detrimental health outcomes. However, advancement to non-alcoholic steatohepatitis (NASH) confers a markedly increased likelihood of developing severe liver pathologies, including fibrosis, cirrhosis, organ failure, and cancer. Although a substantial percentage of NAFLD patients develop NASH, the genetic and molecular mechanisms driving this progression are poorly understood, making it difficult to predict which patients will ultimately develop advanced liver disease. Deficiencies in mechanistic understanding preclude the identification of beneficial prognostic indicators and the development of effective therapies. Mouse models of progressive NAFLD serve as a complementary approach to the direct analysis of human patients. By providing an easily manipulated experimental system that can be rigorously controlled, they facilitate an improved understanding of disease development and progression. In this review, we discuss genetically- and chemically-induced models of NAFLD that progress to NASH, fibrosis, and liver cancer in the context of the major signaling pathways whose disruption has been implicated as a driving force for their development. Additionally, an overview of nutritional models of progressive NAFLD is provided.     

Archaeal diversity in permafrost deposits of Bunger Hills Oasis and King George Island (Antarctica) according to the 16S rRNA gene sequencing

Archaeal communities of permafrost deposits of King George Island and Bunger Hills Oasis (Antarctica) differing in the content of biogenic methane were analyzed using the clone libraries of two 16S rRNA gene regions. Phylotypes belonging to methanogenic archaea were identified in all horizons.     

Adsorption of γ-Aminobutyric acid to phosphatidylserine membranes

The interaction of the negatively-charged phosphatidylserine (PS) and γ-Aminobutyric acid (GABA) is examined in black lipid membranes (BLM) and inverse micelles. GABA does not permeate through PS membranes and, in concentrations of 10^?5-10^?4 M, it reduces the negative potential at the membrane-aqueous solution interface. The effect is owing to the adsorption of the GABA cationic species and the consequent decrease of the negative surface charge density of the membrane. When the intrinsic pH of the membrane-solution interface is considered, the Gouy-Chapman-Stern theory describes the GABA screening effect and makes it possible to calculate the GABA-PS binding constant. This value is compared with that obtained measuring the partition of¹⁴C-GABA between an organic phase containing PS and the aqueous solution. The results presented strongly suggest that the electrostatic force plays a major role in GABA-PS interaction.     

Diversity of cultivable halophilic archaea and bacteria from superficial hypersaline sediments of Tunisian solar salterns

Prokaryotes in the superficial sediments are ecologically important microorganisms that are responsible for the decomposition, mineralization and subsequent recycling of organic matter. The aim of this study was to explore the phylogenetic and functional diversity of halophilic archaea and bacteria isolated from the superficial sediments of solar salterns at Sfax, Tunisia. Sixty four strains were isolated from crystallizer (TS18) and non-crystallizer (M1) ponds and submitted to genotypic characterization and evaluation by amplified ribosomal RNA restriction analysis (ARDRA) techniques. Our findings revealed that the archaeal diversity observed for 29 isolates generated five distinct patterns from the non-crystallizer M1 pond, with Halorubrum chaoviator as the most prevalent cultivable species. However, in the TS18 crystallizer pond, ten restriction patterns were observed, with the prevalence of haloarchaea EB27K, a not yet identified genotype. The construction of a neighbour-joining tree of 16S rRNA gene sequences resulted in the division of the potential new species into two major groups, with four strains closely related to the sequence of the unculturable haloarchaeon EB27K and one strain to the recently described Halovenus aranensis strain. The 35 bacterial strains observed in this work were present only in the non-crystallizer pond (M1) and presented two distinct ARDRA patterns. These strains belonged to the γ-proteobacteria subdivision, with members of Salicola marasensis (83 %) being the most predominant species among the isolates. 16S rRNA gene sequencing revealed that Salicola strains displayed different degrees of homogeneity. The results from pulsed field gel electrophoresis assays showed that the Salicola isolates could be clustered in two distinct groups with different genome sizes.     

Application of Diffusion Growth Chambers for the Cultivation of Marine Sponge-Associated Bacteria

Marine sponges contain dense and diverse microbial communities, which are renowned as a source of bioactive metabolites. The biological activities of sponge-microbe natural products span a broad spectrum, from antibacterial and antifungal to antitumor and antiviral applications. However, the potential of sponge-derived compounds has not been fully realized, due largely to the acknowledged “supply issue.” Most bacteria from environmental samples have resisted cultivation on artificial growth media, and cultivation of sponge-associated bacteria has been a major focus in the search for novel marine natural products. One approach to isolate so-called “uncultivable” microorganisms from different environments is the diffusion growth chamber method. Here, we describe the first application of diffusion growth chambers for the isolation of cultivable and previously uncultivated bacteria from sponges. The study was conducted by implanting diffusion growth chambers in the tissue of Rhabdastrella globostellata reef sponges. In total, 255 16S rRNA gene sequences were obtained, with phylogenetic analyses revealing their affiliations with the Alpha- and Gammaproteobacteria, Bacteroidetes, Actinobacteria, and Firmicutes. Fifteen sequences represented previously uncultivated bacteria belonging to the Bacteroidetes and Proteobacteria (Alpha and Gamma classes). Our results indicate that the diffusion growth chamber approach can be successfully applied in a natural, living marine environment such as sponges.     

Ribosomal RNA contents of maize genotypes with different ribosomal RNA gene numbers

Ribosomal RNA (rRNA) contents were determined in 16 maize genotypes whose individual rRNA gene numbers varied from 5000 to 23,000 per 2C nucleus. Analytical polyacrylamide gel electrophoresis of total RNA showed that no obvious relation existed between rRNA gene number and rRNA content. Only two of nine common inbred lines contained more rRNA than W-23, the inbred with the lowest rRNA gene number. Two of four lines with altered protein content (due to long-term experimental selection) had rRNA contents significantly reduced from those of W-23. A line with an apparent duplication of the nucleolus organizer region of chromosome 6 (called 2-NOR) was expected to possess an elevated quantity of rRNA because it possesses a larger nucleolus; however, we produced a 2-NOR isogenic version and found no difference in rRNA content. The rRNA genes in maize are distributed throughout the NOR-heterochromatin and the NOR-secondary constriction portions of the NOR. The absence of an obvious correlation between rRNA gene number and cellular rRNA content may reflect the presence of a large number of rRNA genes in an inactive state, at least during the stage of growth examined in these experiments.