Fusion induced aggregation of model vesicles studied by dynamic and static light scattering

Membrane fusion is a key step in the virus mediated cell fusion. The vesicular dispersion serves as a model system to study the membrane fusion. We employed dynamic and static light scattering to study the fusion of phosphatidylcholine vesicles in the presence of model fusion peptide fragments from the hemagglutinin HA2 protein. The fusion-induced aggregation under the present experimental setup exhibited strong pH dependence, similar to the parental viral protein. Replacement of the glycine residue at the extreme amino terminus by glutamic acid (G1E) abolished fusion activity. The average molecular mass and diameter of vesicular dispersion obtained from static and dynamic light scattering measurements respectively at neutral and acidic pH showed about three fold increase in acidic solution containing wild type fusion peptide. The light scattering data are consistent with lipid mixing results. The present work demonstrates the utility of light scattering as a facile means to monitor the fusion process.     

Macromolecular mimicry in translation initiation: a model for the initiation factor IF2 on the ribosome

Protein biosynthesis in bacteria is controlled by a number of translation factors. Recent data based on comparison of sequence and structure data of translation factors have established a novel hypothesis for their interaction with the ribosome: initiation, elongation, and termination factors may use a common or partly overlapping binding site on the ribosome in a process of macromolecular mimicry of an A-site-bound tRNA. This paper reviews structural knowledge and tRNA macromolecular mimicry involvement of translation initiation factor IF2. Furthermore, a model is proposed for the factor and its interaction with the ribosome during the formation of the translation initiation complex.     

Poliovirus translation: a paradigm for a novel initiation mechanism

All eukaryotic cellular mRNAs, and most viral mRNAs, are blocked at their 5' ends with a cap structure (m7GpppX, where X is any nucleotide). Poliovirus, along with a small number of other animal and plant viral mRNAs, does not contain a 5' cap structure. Since the cap structure functions to facilitate ribosome binding to mRNA, translation of polio-virus must proceed by a cap-independent mechanism. Consistent with this, recent studies have shown that ribosomes can bind to an internal region within the long 5' noncoding sequence of poliovirus RNA. Possible mechanisms for cap-independent translation are discussed. Cap-independent translation of poliovirus RNA is of major importance to the mechanism of shut-off of host protein synthesis after infection. Moreover, it is likely to play a role in determining poliovirus neurovirulence and attenuation.     

Selective stimulation of translation of leaderless mRNA by initiation factor 2: evolutionary implications for translation

Translation initiation in bacteria involves a stochastic binding mechanism in which the 30S ribosomal subunit first binds either to mRNA or to initiator tRNA, fMet-tRNA(f)(Met). Leaderless lambda cI mRNA did not form a binary complex with 30S ribosomes, which argues against the view that ribosomal recruitment signals other than a 5'-terminal start codon are essential for translation initiation of these mRNAs. We show that, in Escherichia coli, translation initiation factor 2 (IF2) selectively stimulates translation of lambda cI mRNA in vivo and in vitro. These experiments suggest that the start codon of leaderless mRNAs is recognized by a 30S-fMet-tRNA(f)(Met)-IF2 complex, an intermediate equivalent to that obligatorily formed during translation initiation in eukaryotes. We further show that leaderless lambda cI mRNA is faithfully translated in vitro in both archaebacterial and eukaryotic translation systems. This suggests that translation of leaderless mRNAs reflects a fundamental capability of the translational apparatus of all three domains of life and lends support to the hypothesis that the translation initiation pathway is universally conserved.     

TICO: a tool for improving predictions of prokaryotic translation initiation sites

SUMMARY: We provide the tool 'TICO' (Translation Initiation site COrrection) for improving the results of conventional gene finders for prokaryotic genomes with regard to exact localization of the translation initiation site (TIS). At the current state TICO provides an interface for direct post processing of the predictions obtained from the widely used program GLIMMER. Our program is based on a clustering algorithm for completely unsupervised scoring of potential TIS locations. AVAILABILITY: Our tool can be freely accessed through a web interface at http://tico.gobics.de/ CONTACT: maike@gobics.de     

Hon-yaku: a biology-driven Bayesian methodology for identifying translation initiation sites in prokaryotes

Background  

Computational prediction methods are currently used to identify genes in prokaryote genomes. However, identification of the correct translation initiation sites remains a difficult task. Accurate translation initiation sites (TISs) are important not only for the annotation of unknown proteins but also for the prediction of operons, promoters, and small non-coding RNA genes, as this typically makes use of the intergenic distance. A further problem is that most existing methods are optimized for Escherichia coli data sets; applying these methods to newly sequenced bacterial genomes may not result in an equivalent level of accuracy.     

Cap- and initiator tRNA-dependent initiation of TYMV polyprotein synthesis by ribosomes: evaluation of the Trojan horse model for TYMV RNA translation

Turnip yellow mosaic virus (TYMV) RNA directs the translation of two overlapping open reading frames. Competing models have been previously published to explain ribosome access to the downstream polyprotein cistron. The Trojan horse model, based on cell-free experiments, proposes noncanonical cap-independent initiation in which the 3'-terminal tRNA-like structure (TLS) functionally replaces initiator tRNA, and the valine bound to the TLS becomes cis-incorporated into viral protein. The initiation coupling model, based on in vivo expression and ribosome toe-printing studies, proposes a variation of canonical leaky scanning. Here, we have re-examined the wheat germ extract experiments that led to the Trojan horse model, incorporating a variety of controls. We report that (1) translation in vitro from the polyprotein AUG of TYMV RNA is unchanged after removal of the 3' TLS but is stimulated by the presence of a 5'-cap; (2) the presence of free cap analog or edeine (which interferes with initiation at the ribosomal P site and its tRNA(i) (Met) involvement) inhibits translation from the polyprotein AUG; (3) the toe-prints of immediately post-initiation ribosomes on TYMV RNA are similar with and without an intact TLS; and (4) significant deacylation of valyl-TYMV RNA in wheat germ extract can complicate the detection of cis-incorporation. These results favor the initiation coupling model.     

Odor and irritation thresholds for ammonia: a comparison between static and dynamic olfactometry

Odor and lateralization (irritation) thresholds (LTs) for ammonia vapor were measured using static and dynamic olfactometry. The purpose of the study was to explore the test-retest reliability and comparability of dynamic olfactometry methodology, generally used to determine odor thresholds following European Committee for Standardization guidelines in the context of odor regulations to outside emissions, with static olfactometry. Within a 2-week period, odor and LTs for ammonia were obtained twice for each method for 24 females. No significant differences between methods were found: mean odor detection thresholds (ODTs) were 2.6 parts per million (ppm) for either method (P = 0.96), and mean LTs were 31.7 and 60.9 ppm for the static and dynamic method, respectively (P = 0.07). Test-retest reliability was higher for the dynamic than for the static method (r = 0.61 vs. 0.14 for ODTs and r = 0.86 vs. 0.45 for LTs). The choice of optimal method for any application, however, depends not only on psychometric factors but also on practical factors such as physicochemical properties of the compound, availability of equipment and expertise, task efficiency, and costs.     

Immunogenic tumor cell death induced by chemoradiotherapy: molecular mechanisms and a clinical translation

Chemoradiotherapy can induce immunogenic cell death, triggering danger signals such as high-mobility group box 1 protein, and resulting in T-cell immunity. This concept can potentially be harnessed for clinical therapy to enhance tumor-specific immunity. There is however limited information to translate this theory directly in a clinical setting. In this review, we will discuss and summarize molecular and cellular mechanisms underlying immunogenic tumor cell death induced by chemoradiotherapy, with emphasis on a clinical translation.     

Regulation of senescence by eukaryotic translation initiation factor 5A: implications for plant growth and development

Regulation of protein synthesis is increasingly being recognized as an important determinant of cell proliferation and senescence. In particular, recent evidence indicates that eukaryotic translation initiation factor 5A (eIF-A) plays a pivotal role in this determination. Separate isoforms of eIF-5A appear to facilitate the translation of mRNAs required for cell division and cell death. This raises the possibility that eIF-5A isoforms are elements of a biological switch that is in one position in dividing cells and in another position in dying cells. Changes in the position of this putative switch in response to physiological and environmental cues are likely to have a significant impact on plant growth and development.     

Realizing the promise of population biobanks: a new model for translation

The promise of science lies in expectations of its benefits to societies and is matched by expectations of the realisation of the significant public investment in that science. In this paper, we undertake a methodological analysis of the science of biobanking and a sociological analysis of translational research in relation to biobanking. Part of global and local endeavours to translate raw biomedical evidence into practice, biobanks aim to provide a platform for generating new scientific knowledge to inform development of new policies, systems and interventions to enhance the public’s health. Effectively translating scientific knowledge into routine practice, however, involves more than good science. Although biobanks undoubtedly provide a fundamental resource for both clinical and public health practice, their potentiating ontology—that their outputs are perpetually a promise of scientific knowledge generation—renders translation rather less straightforward than drug discovery and treatment implementation. Biobanking science, therefore, provides a perfect counterpoint against which to test the bounds of translational research. We argue that translational research is a contextual and cumulative process: one that is necessarily dynamic and interactive and involves multiple actors. We propose a new multidimensional model of translational research which enables us to imagine a new paradigm: one that takes us from bench to bedside to backyard and beyond, that is, attentive to the social and political context of translational science, and is cognisant of all the players in that process be they researchers, health professionals, policy makers, industry representatives, members of the public or research participants, amongst others.     

Generating compatible translation initiation regions for heterologous gene expression in Escherichia coli by exhaustive periShine-Dalgarno mutagenesis. Human glutathione reductase cDNA as a model.

Adaptation of eucaryotic cDNA to heterologous expression was studied by mutating the translation initiation (TI) region upstream (mTI) and downstream (MTI) of the start codon. In the mTI subregion the 8 bases flanking the invariant Shine-Dalgarno motif GG-AG were mutagenized exhaustively, while the MTI subregion was subjected to random silent mutations at the wobble positions. The quality of a given TI sequence was judged on the basis of expressed enzyme activity. Low-yield and high-yield mutants of both TI subregions were selected and recombined systematically. The analysis of these double cartridges gave the following results: 1. As a rule, an unfavourable MTI subregion can be compensated for by mutations in the mTI subregion and vice versa. 2. The compatibility between mTI and MTI subregion is explainable at least in part by a low interaction tendency; a delta G(o)'-value of -10.7 kcal/mol appears to be a physical threshold for heterologous cDNA expression. 3. On the basis of periShine-Dalgarno mutations, the expression yield for different cDNA sequences could be increased by 1 to 2 orders of magnitude. One of these sequences encoded delta(1-15)human glutathione reductase, a mutant lacking the flexible N-terminal extension of the protein. In conclusion, to study and overcome TI region-based expression problems it is worthwhile to start out with a versatile vector containing exhaustive mutations in the periShine-Dalgarno sequences; as a rule the coding MTI subregion can be kept unchanged.     

Extracellular neural microstimulation may activate much larger regions than expected by simulations: a combined experimental and modeling study

Electrical stimulation of the central nervous system has been widely used for decades for either fundamental research purposes or clinical treatment applications. Yet, very little is known regarding the spatial extent of an electrical stimulation. If pioneering experimental studies reported that activation threshold currents (TCs) increase with the square of the neuron-to-electrode distance over a few hundreds of microns, there is no evidence that this quadratic law remains valid for larger distances. Moreover, nowadays, numerical simulation approaches have supplanted experimental studies for estimating TCs. However, model predictions have not yet been validated directly with experiments within a common paradigm. Here, we present a direct comparison between experimental determination and modeling prediction of TCs up to distances of several millimeters. First, we combined patch-clamp recording and microelectrode array stimulation in whole embryonic mouse spinal cords to determine TCs. Experimental thresholds did not follow a quadratic law beyond 1 millimeter, but rather tended to remain constant for distances larger than 1 millimeter. We next built a combined finite element - compartment model of the same experimental paradigm to predict TCs. While theoretical TCs closely matched experimental TCs for distances <250 microns, they were highly overestimated for larger distances. This discrepancy remained even after modifications of the finite element model of the potential field, taking into account anisotropic, heterogeneous or dielectric properties of the tissue. In conclusion, these results show that quadratic evolution of TCs does not always hold for large distances between the electrode and the neuron and that classical models may underestimate volumes of tissue activated by electrical stimulation.     

Biological control of thrips and whiteflies by a shared predator: Two pests are better than one

We studied the capacity of one species of predator to control two major pests of greenhouse crops, Western flower thrips (Frankliniella occidentalis (Pergande)) and the greenhouse whitefly (Trialeurodes vaporariorum (Westwood)). In such a one-predator–two-prey system, indirect interactions can occur between the two pest species, such as apparent competition and apparent mutualism. Whereas apparent competition is desired because it brings pest levels down, apparent mutualism is not, because it does the opposite. Because apparent competition and apparent mutualism occurs at different time scales, it is important to investigate the effects of a shared natural enemy on biological control on a time scale relevant for crop growth. We evaluated the control efficacy of the predatory mites Amblyseius swirskii (Athias-Henriot) and Euseius ovalis (Evans) in cucumber crops in greenhouse compartments with only thrips, only whiteflies or both herbivorous insects together. Each of the two predators controlled thrips, but A. swirskii reduced thrips densities the most. There was no effect of the presence of whiteflies on thrips densities. Whitefly control by each of the two predators in absence of thrips was not sufficient, yet better with E. ovalis. However, whitefly densities in presence of thrips were reduced dramatically, especially by A. swirskii. The densities of predators were up to 15 times higher in presence of both pests than in the single-pest treatments. Laboratory experiments with A. swirskii suggest that this is due to a higher juvenile survival and developmental rate on a mixed diet. Hence, better control may be achieved not only because of apparent competition, but also through a positive effect of mixed diets on predator population growth. This latter phenomenon deserves more attention in experimental and theoretical work on biological control and apparent competition.     

Activation of p53 as a causal step for atherosclerosis induced by polycyclic aromatic hydrocarbons

This study was performed to prove our hypothesis that the metabolite(s) of polycyclic aromatic hydrocarbons (PAHs) caused the activation or phosphorylation of p53 via DNA damage to suppress the liver X receptor (LXR)-mediated signal transductions as a probably more direct mechanism. We found that LXR-mediated trans-activation was inhibited by 3-methylchoranthrene (MC) and doxorubicin (Dox) in HepG2 cells carrying wild-type p53, but not in Hep3B cells possessing mutant p53. The exogenous expression of wild-type p53 suppressed the LXR-mediated trans-activation in Hep3B cells. The expression of mRNA for ATP binding cassette A1 was suppressed by MC and Dox in HepG2 cells. The protein expression of retinoid X receptor (RXR), a partner of LXR to form a heterodimer, was suppressed by MC and Dox in HepG2 cells.