mRNA pseudoknot structures can act as ribosomal roadblocks

Several viruses utilize programmed ribosomal frameshifting mediated by mRNA pseudoknots in combination with a slippery sequence to produce a well defined stochiometric ratio of the upstream encoded to the downstream-encoded protein. A correlation between the mechanical strength of mRNA pseudoknots and frameshifting efficiency has previously been found; however, the physical mechanism behind frameshifting still remains to be fully understood. In this study, we utilized synthetic sequences predicted to form mRNA pseudoknot-like structures. Surprisingly, the structures predicted to be strongest lead only to limited frameshifting. Two-dimensional gel electrophoresis of pulse labelled proteins revealed that a significant fraction of the ribosomes were frameshifted but unable to pass the pseudoknot-like structures. Hence, pseudoknots can act as ribosomal roadblocks, prohibiting a significant fraction of the frameshifted ribosomes from reaching the downstream stop codon. The stronger the pseudoknot the larger the frameshifting efficiency and the larger its roadblocking effect. The maximal amount of full-length frameshifted product is produced from a structure where those two effects are balanced. Taking ribosomal roadblocking into account is a prerequisite for formulating correct frameshifting hypotheses.     

Indole can act as an extracellular signal to regulate biofilm formation of Escherichia coli and other indole-producing bacteria

We demonstrated previously that genetic inactivation of tryptophanase is responsible for a dramatic decrease in biofilm formation in the laboratory strain Escherichia coli S17-1. In the present study, we tested whether the biochemical inhibition of tryptophanase, with the competitive inhibitor oxindolyl-L-alanine, could affect polystyrene colonization by E. coli and other indole-producing bacteria. Oxindolyl-L-alanine inhibits, in a dose-dependent manner, indole production and biofilm formation by strain S17-1 grown in Luria-Bertani (LB) medium. Supplementation with indole at physiologically relevant concentrations restores biofilm formation by strain S17-1 in the presence of oxindolyl-L-alanine and by mutant strain E. coli 3714 (S17-1 tnaA::Tn5) in LB medium. Oxindolyl-L-alanine also inhibits the adherence of S17-1 cells to polystyrene for a 3-h incubation time, but mutant strain 3714 cells are unaffected. At 0.5 mg/mL, oxindolyl-L-alanine exhibits inhibitory activity against biofilm formation in LB medium and in synthetic urine for several clinical isolates of E. coli, Klebsiella oxytoca, Citrobacter koseri, Providencia stuartii, and Morganella morganii but has no affect on indole-negative Klebsiella pneumoniae strains. In conclusion, these data suggest that indole, produced by the action of tryptophanase, is involved in polystyrene colonization by several indole-producing bacterial species. Indole may act as a signalling molecule to regulate the expression of adhesion and biofilm-promoting factors.     

Hairpin ribozyme-antisense RNA constructs can act as molecular Lassos

We have developed a novel class of antisense agents, RNA Lassos, which are capable of binding to and circularizing around complementary target RNAs. The RNA Lasso consists of a fixed sequence derived from the hairpin ribozyme and an antisense segment whose size and sequence can be varied to base pair with accessible sites in the target RNA. The ribozyme catalyzes self-processing of the 5'- and 3'-ends of a transcribed Lasso precursor and ligates the processed ends to produce a circular RNA. The circular and linear forms of the self-processed Lasso coexist in an equilibrium that is dependent on both the Lasso sequence and the solution conditions. Lassos form strong, noncovalent complexes with linear target RNAs and form true topological linkages with circular targets. Lasso complexes with linear RNA targets were detected by denaturing gel electrophoresis and were found to be more stable than ordinary RNA duplexes. We show that expression of a fusion mRNA consisting of a sequence from the murine tumor necrosis factor-alpha (TNF-alpha) gene linked to luciferase reporter can be specifically and efficiently blocked by an anti-TNF Lasso. We also show in cell culture experiments that Lassos directed against Fas pre-mRNA were able to induce a change in alternative splicing patterns.     

Lipopolysaccharides from Helicobacter pylori can act as antagonists for Toll-like receptor 4

Infection with Helicobacter pylori, a Gram-negative bacterium, is strongly associated with gastric ulcers and adenocarcinoma. The mechanisms by which the innate immune system recognizes H. pylori lipopolysaccharide (LPS) remain unclear. Contradictory reports exist that suggest that Toll-like receptors are involved. In this study we evaluated the interactions of Toll-like receptors with LPS from different strains of H. pylori. Using reporter cell lines, as well as HEK293 cells transfected with either CD14 and TLR4, or CD14 and TLR2, we show that H. pylori LPS-induced cell activation is mediated through TLR2. In addition, for the first time, we report that LPS from some H. pylori strains are able to antagonize TLR4. The antagonistic activity of H. pylori LPS from certain strains, as well as the activation via TLR2, might give H. pylori an advantage over the host that may be associated with the clinical outcome of H. pylori infection.     

Imatinib can act as an Allosteric Activator of Abl Kinase

Imatinib is an ATP-competitive inhibitor of Bcr-Abl kinase and the first drug approved for chronic myelogenous leukemia (CML) treatment. Here we show that imatinib binds to a secondary, allosteric site located in the myristoyl pocket of Abl to function as an activator of the kinase activity. Abl transitions between an assembled, inhibited state and an extended, activated state. The equilibrium is regulated by the conformation of the αΙ helix, which is located nearby the allosteric pocket. Imatinib binding to the allosteric pocket elicits an αΙ helix conformation that is not compatible with the assembled state, thereby promoting the extended state and stimulating the kinase activity. Although in wild-type Abl the catalytic pocket has a much higher affinity for imatinib than the allosteric pocket does, the two binding affinities are comparable in Abl variants carrying imatinib-resistant mutations in the catalytic site. A previously isolated imatinib-resistant mutation in patients appears to be mediating its function by increasing the affinity of imatinib for the allosteric pocket, providing a hitherto unknown mechanism of drug resistance. Our results highlight the benefit of combining imatinib with allosteric inhibitors to maximize their inhibitory effect on Bcr-Abl.     

The mycobacterial thioredoxin peroxidase can act as a one-cysteine peroxiredoxin

Thioredoxin peroxidase (TPx) has been reported to dominate the defense against H(2)O(2), other hydroperoxides, and peroxynitrite at the expense of thioredoxin (Trx) B and C in Mycobacterium tuberculosis (Mt). By homology, the enzyme has been classified as an atypical 2-C-peroxiredoxin (Prx), with Cys(60) as the "peroxidatic" cysteine (C(P)) forming a complex catalytic center with Cys(93) as the "resolving" cysteine (C(R)). Site-directed mutagenesis confirms Cys(60) to be C(P) and Cys(80) to be catalytically irrelevant. Replacing Cys(93) with serine leads to fast inactivation as seen by conventional activity determination, which is associated with oxidation of Cys(60) to a sulfinic acid derivative. However, in comparative stopped-flow analysis, WT-MtTPx and MtTPx C93S reduce peroxynitrite and react with TrxB and -C similarly fast. Reduction of pre-oxidized WT-MtTPx and MtTPx C93S by MtTrxB is demonstrated by monitoring the redox-dependent tryptophan fluorescence of MtTrxB. Furthermore, MtTPx C93S remains stable for 10 min at a morpholinosydnonimine hydrochloride-generated low flux of peroxynitrite and excess MtTrxB in a dihydrorhodamine oxidation model. Liquid chromatography-tandem mass spectrometry analysis revealed disulfide bridges between Cys(60) and Cys(93) and between Cys(60) and Cys(80) in oxidized WT-MtTPx. Reaction of pre-oxidized WT-MtTPx and MtTPx C93S with MtTrxB C34S or MtTrxC C40S yielded dead-end intermediates in which the Trx mutants are preferentially linked via disulfide bonds to Cys(60) and never to Cys(93) of the TPx. It is concluded that neither Cys(80) nor Cys(93) is required for the catalytic cycle of the peroxidase. Instead, MtTPx can react as a 1-C-Prx with Cys(60) being the site of attack for both the oxidizing and the reducing substrate. The role of Cys(93) is likely to conserve the oxidation equivalents of the sulfenic acid state of C(P) as a disulfide bond to prevent overoxidation of Cys(60) under a restricted supply of reducing substrate.     

Quartz fibers as templates for biopolymers

The polymerization of silica in water solution to form quartz fibers proceeds by a dehydration process, analogous to condensation polymerization in organic high-polymers, in which monomeric Si(OH)₄ groups unite through Si–O–Si bonds with the elimination of H₂O. The resulting fibers are structurally polar along the direction of elongation, are enantiomorphous, and generally shown stereospecific twisting around the direction of elongation. In these regards the fibers are analogues of biopolymers such as RNA and DNA. Quartz also possesses specific adsorptive relations to a wide range of organic substances including monomer amino acids, short-chain polypeptides, and proteins. These involve hydrogen-bonding between (OH) or silanoi groups on the surface of the quartz with active side-groups on the organic molecules and in part are epitaxial through dimensional coincidences in the interface.Geochemical evidence indicates that quartz was deposited in the early Precambrian ocean either by direct crystallization from seawater or by recrystallization of amorphous silica. What is of interest is the possible role of quartz fibers as a template and co-polymer in the passage of biomonomers in the pre-biotic ocean to the long-chain biopolymers such as nucleic acids and proteins that are involved in life processes.     

Woodpeckers can act as dispersal vectors for fungi,plants, and microorganisms

Bird‐mediated dispersal is presumed to be important in the dissemination of many different types of organisms, but concrete evidence remains scarce. This is especially true for biota producing microscopic propagules. Tree‐dwelling birds, such as woodpeckers, would seem to represent ideal dispersal vectors for organisms growing on standing tree trunks such as epiphytic lichens and fungi. Here, we utilize bird natural history collections as a novel source of data for studying dispersal ecology of plants, fungi, and microorganisms. We screened freshly preserved specimens of three Finnish woodpecker species for microscopic propagules. Samples were taken from bird feet, and chest and tail feathers. Propagules were extracted using a sonication–centrifugation protocol, and the material obtained was studied using light microscopy. Diverse biological material was recovered from all specimens of all bird species, from all positions sampled. Most abundant categories of discovered biological material included bryophyte fragments, fungal spores, and vegetative propagules of lichens. Also, freshwater diatoms, bryophyte spores, algal cells, testate amebae, rotifers, nematodes, pollen, and insect scales were identified. The method developed here is applicable to living specimens as well, making it a versatile tool for further research. Our findings highlight the potential of bird‐mediated dispersal for diverse organisms and showcase the use of natural history collections in ecological research.     

The site of RanGTP generation can act as an organizational cue for mitotic microtubules

Background information. RanGTP, which is generated on chromosomes during mitosis, is required for microtubule spindle assembly. Due to its restricted spatial generation within the cell it has been suggested that RanGTP acts as a spatial cue to organize site‐specific spindle assembly within the cell. However, the absence of a detectable sharp gradient of RanGTP in somatic cells has led to suggestions that it may only act as a spatial cue in large cells and that it may operate as a general activator of the mitotic cytosol in somatic cells. Results. We report that ectopic generation of RanGTP at the plasma membrane stimulates the formation of organized arrays of microtubules at the plasma membrane. Conclusions. These results suggest that the site of RanGTP generation in a mitotic somatic cell can generate critical spatial information that specifies where microtubules grow towards and where microtubules are organized. As RanGTP is normally generated on chromosomes, these results suggest that RanGTP may play an important role in specifying that spindle assembly occurs around chromosomes.     

Pro-inflammatory cytokines can act as intracellular modulators of commensal bacterial virulence

Interactions between commensal pathogens and hosts are critical for disease development but the underlying mechanisms for switching between the commensal and virulent states are unknown. We show that the human pathogen Neisseria meningitidis, the leading cause of pyogenic meningitis, can modulate gene expression via uptake of host pro-inflammatory cytokines leading to increased virulence. This uptake is mediated by type IV pili (Tfp) and reliant on the PilT ATPase activity. Two Tfp subunits, PilE and PilQ, are identified as the ligands for TNF-α and IL-8 in a glycan-dependent manner, and their deletion results in decreased virulence and increased survival in a mouse model. We propose a novel mechanism by which pathogens use the twitching motility mode of the Tfp machinery for sensing and importing host elicitors, aligning with the inflamed environment and switching to the virulent state.     

The tsx protein of Escherichia coli can act as a pore for amino acids

The tsx protein is known to be a specific diffusion pathway for nucleosides. The ability of this protein to facilitate the transport of molecules other than nucleosides was examined in strains lacking detectable amounts of porin (ompB mutants). The tsx protein was shown to promote serine, glycine, and phenylalanine transport and to have no effect on either glucose or arginine transport.     

The p53 proto-oncogene can act as a suppressor of transformation

DNA clones of the wild-type p53 proto-oncogene inhibit the ability of E1A plus ras or mutant p53 plus ras-activated oncogenes to transform primary rat embryo fibroblasts. The rare clones of transformed foci that result from E1A plus ras plus wild-type p53 triple transfections all contain the p53 DNA in their genome, but the great majority fail to express the p53 protein. The three cell lines derived from such foci that express p53 all produce mutant p53 proteins with properties similar or identical to transformation-activated p53 proteins. The p53 mutants selected in this fashion (transformation in vitro) resemble the p53 mutants selected in tumors (in vivo). These results suggest that the p53 proto-oncogene can act negatively to block transformation.     

Capacity of aquatic bacteria to act as recipients of plasmid DNA

A total of 68 gram-negative freshwater bacterial isolates were screened for their ability to receive and express plasmids from Pseudomonas aeruginosa donors. The plate mating technique identified 26 of the isolates as recipient active for the self-transmissible wide-host-range plasmid R68; 10 were recipient active by R68 mobilization for the wide-host-range plasmid cloning vector R1162. Frequencies of transfer were compared by using three conjugal transfer procedures: broth, plate, and filter mating. For every recipient tested, a solid environment was superior to a liquid environment for transfer. The broth mating technique failed to demonstrate R68 transfer in 63% of the recipient-active isolates. Filter mating, in general, yielded the highest transfer frequencies. The more-rapid plate mating procedure, however, was just as sensitive for testing the capacity of natural isolates to participate in conjugal plasmid transfer.     

River dolphins can act as population trend indicators in degraded freshwater systems

Conservation attention on charismatic large vertebrates such as dolphins is often supported by the suggestion that these species represent surrogates for wider biodiversity, or act as indicators of ecosystem health. However, their capacity to act as indicators of patterns or trends in regional biodiversity has rarely been tested. An extensive new dataset of >300 last-sighting records for the Yangtze River dolphin or baiji and two formerly economically important fishes, the Yangtze paddlefish and Reeves' shad, all of which are probably now extinct in the Yangtze, was collected during an interview survey of fishing communities across the middle-lower Yangtze drainage. Untransformed last-sighting date frequency distributions for these species show similar decline curves over time, and the linear gradients of transformed last-sighting date series are not significantly different from each other, demonstrating that these species experienced correlated population declines in both timing and rate of decline. Whereas species may be expected to respond differently at the population level even in highly degraded ecosystems, highly vulnerable (e.g. migratory) species can therefore display very similar responses to extrinsic threats, even if they represent otherwise very different taxonomic, biological and ecological groupings. Monitoring the status of river dolphins or other megafauna therefore has the potential to provide wider information on the status of other threatened components of sympatric freshwater biotas, and so represents a potentially important monitoring tool for conservation management. We also show that interview surveys can provide robust quantitative data on relative population dynamics of different species.     

Actin can act as a cofactor for a viral proteinase in the cleavage of the cytoskeleton

Cytoskeletal proteins are exploited by many viruses during infection. We report a novel finding that actin can act as a cofactor for the adenovirus proteinase (AVP) in the degradation of cytoskeletal proteins. Transfection studies in HeLa cells revealed AVP localized with cytokeratin 18, and this was followed by destruction of the cytokeratin network. For AVP to cleave cytokeratin 18, a cellular cofactor was shown to be required, consistent with AVP being synthesized as an inactive proteinase. Actin was considered a cellular cofactor for AVP, because the C terminus of actin is homologous to a viral cofactor for AVP. AVP was shown to bind to the C terminus of actin, and in doing so AVP exhibited full enzymatic activity. In vitro, actin was a cofactor in the cleavage of cytokeratin 18 by AVP. The proteinase alone could not cleave cytokeratin 18, but in the presence of actin, AVP cleaved cytokeratin 18. Indeed, actin itself was shown to be a cofactor and a substrate for its own destruction in that it was cleaved by AVP in vitro. Cleavage of cytoskeletal proteins weakens the structure of the cell, and therefore, actin as a cofactor may play a role in cell lysis and release of nascent virions.     

Quartz fibers as templates for biopolymers.

The polymerization of silica in water solution to form quartz fibers proceeds by a dehydration process, analogous to condensation polymerization in organic high-polymers, in which monomeric Si(OH)4 groups unite through Si--O--Si bonds with the elimination of H2O. The resulting fibers are structurally polar along the direction of elongation, are enantiomorphous, and generally show stereospecific twisting around the direction of elongation. In these regards the fibers are analogues of biopolymers such as RNA and DNA. Quartz also possesses specific adsorptive relations to a wide range of organic substances including monomer amino acids, short-chain polypeptides, and proteins. These involve hydrogen-bonding between (OH) or silanol groups on the surface of the quartz with active side-groups on the organic molecules, and in part are epitaxial through dimensional coincidences in the interface. Geochemical evidence indicates that quartz was deposited in the early Precambrian ocean either by direct crystallization from seawater or by recrystallization of amorphous silica. What is of interest is the possible role of quartz fibers as a template and co-polymer in the passage of biomonomers in the pre-biotic ocean to the long-chain biopolymers such as nucleic acids and proteins that are involved in life processes.