Surprisingly fast disappearance of beta-lactam selection pressure in cultivation as detected with novel biosensing approaches

Tetracycline and beta-lactam resistances among others are used as selection markers in the production of recombinant proteins. The beta-lactam resistance is based on degradation, i.e. the selection pressure gradually disappears from the culture, whereas tetracycline resistance is based on active efflux. We have studied the kinetics of the stability of antibiotic selection pressure in culture using a simple model system (pBR322 in Escherichia coli). Concentrations of ampicillin, carbenicillin and tetracycline were measured with novel sensor cells developed in our lab. These cells are specifically induced to produce light in the presence of the drugs and here their performance was shown to be excellent in monitoring antibiotic concentrations in cell culture. The sensor cells are cheap to produce and use and a high number of samples can be analysed simultaneously. To our surprise, ampicillin and carbenicillin were completely degraded after 2.5-3.0 h of culture, although it has been widely claimed that especially carbenicillin is a good selective agent, whereas tetracycline was stable in culture. beta-lactamase activity in culture was found to correlate with the kinetics of ampicillin degradation.     

Dynamic covariation between gene expression and genome characteristics

Gene and protein expression is controlled so that cells can react to changing intra- and extracellular signals by modulating biochemical networks and pathways. We have previously shown that gene expression and the properties of expressed proteins are dynamically correlated. Here we investigated correlations between gene related parameters and gene expression patterns, and found statistically significant correlations in microarray datasets for different cell types, organisms and processes, including human B and T cell stimulation, cell cycle in HeLa cells, infection in intestinal epithelial cells, Drosophila melanogaster life span, and Saccharomyces cerevisiae cell cycle. Our method was applied to time course datasets individually for each time point. We derived from sequence information numerous parameters for nucleotide composition, two-base composition, codon usage, skew parameters, and codon bias. In addition to coding regions, we also investigated correlations for complete genes and introns. Significant dynamic correlations were identified for each of the analyses. Our method also proved useful for detecting dynamic shifts in gene expression profiles, such as in the D. melanogaster dataset. Detection of changes in the properties of expressed genes and proteins might be useful for predicting or following biological processes, responses, growth, differentiation and possibly in related disorders.     

Virus-induced gene silencing for Asteraceae-a reverse genetics approach for functional genomics in Gerbera hybrida

Virus-induced gene silencing (VIGS) is a natural defence mechanism in plants which leads to sequence-specific degradation of viral RNA. For identifying gene functions, Tobacco rattle virus (TRV)-based VIGS has been applied for silencing of endogenous genes in many plant species. Gerbera hybrida (Asteraceae) has emerged as a novel model for studies in flower development and secondary metabolism. For this highly heterozygous species, functional studies have been conducted through reverse genetic methods by producing stable transgenic lines, which, however, is labour-intensive and time-consuming. For the development of TRV-based VIGS system for gerbera, and for the first time for an Asteraceaeous species, we screened several gerbera cultivars and optimized the agroinfiltration methods for efficient silencing. Gene fragments for gerbera phytoene desaturase (GPDS) and Mg-chelatase subunits (GChl-H and GChl-I), expressed from a TRV vector, induced silencing phenotypes in leaves, scapes, and involucral bracts indicating their feasibility as markers for green tissues. In addition, robust silencing symptoms were achieved in gerbera floral tissues by silencing the anthocyanin pathway gene for chalcone synthase (GCHS1) and a gerbera B-type MADS-box gene globosa (GGLO1), confirming the phenotypes previously observed in stable transgenic lines. Unexpectedly, photobleaching induced by GPDS and GChl-H or GChl-I silencing, or by the herbicide norflurazon, resulted in silencing of the polyketide synthase gene G2PS1, which has no apparent connections to carotenoid or chlorophyll biosynthesis. We have shown feasibility of VIGS for functional studies in gerbera, but our results also show that selection of the marker gene for silencing must be critically evaluated.     

Pursuit Eye-Movements in Curve Driving Differentiate between Future Path and Tangent Point Models

For nearly 20 years, looking at the tangent point on the road edge has been prominent in models of visual orientation in curve driving. It is the most common interpretation of the commonly observed pattern of car drivers looking through a bend, or at the apex of the curve. Indeed, in the visual science literature, visual orientation towards the inside of a bend has become known as “tangent point orientation”. Yet, it remains to be empirically established whether it is the tangent point the drivers are looking at, or whether some other reference point on the road surface, or several reference points, are being targeted in addition to, or instead of, the tangent point. Recently discovered optokinetic pursuit eye-movements during curve driving can provide complementary evidence over and above traditional gaze-position measures. This paper presents the first detailed quantitative analysis of pursuit eye movements elicited by curvilinear optic flow in real driving. The data implicates the far zone beyond the tangent point as an important gaze target area during steady-state cornering. This is in line with the future path steering models, but difficult to reconcile with any pure tangent point steering model. We conclude that the tangent point steering models do not provide a general explanation of eye movement and steering during a curve driving sequence and cannot be considered uncritically as the default interpretation when the gaze position distribution is observed to be situated in the region of the curve apex.     

Coxsackievirus B3-induced cellular protrusions: structural characteristics and functional competence

Virus-induced alterations in cell morphology play important roles in the viral life cycle. To examine the intracellular events of coxsackievirus B3 (CVB3) infection, green monkey kidney (GMK) cells were either inoculated with the virus or transfected with the viral RNA. Various microscopic and flow cytometric approaches demonstrated the emergence of CVB3 capsid proteins at 8 h posttransfection, followed by morphological transformation of the cells. The morphological changes included formation of membranous protrusions containing viral capsids, together with microtubules and actin. Translocation of viral capsids into these protrusions was sensitive to cytochalasin D, suggesting the importance of actin in the process. Three-dimensional (3D) live-cell imaging demonstrated frequent contacts between cellular protrusions and adjacent cells. Markedly, in spite of an increase in the cellular viral protein content starting 8 h postinfection, no significant decrease in cell viability or increase in the amount of early apoptotic markers was observed by flow cytometry by 28 h postinfection. Comicroinjection of viral RNA and fluorescent dextran in the presence of neutralizing virus antibody suggested that these protrusions mediated the spread of infection from one cell to another prior to virus-induced cell lysis. Altogether, the CVB3-induced cellular protrusions could function as a hitherto-unknown nonlytic mechanism of cell-to-cell transmission exploited by enteroviruses.     

A carbon cost-gain model explains the observed patterns of xylem safety and efficiency

Efficient water transport from the soil to the leaves is essential for plant function, while building and maintaining the water transport structure in the xylem require a major proportion of the assimilated carbon of the tree. Xylem transport also faces additional challenges as water in the xylem is under tension and therefore cavitation cannot be completely avoided. We constructed a model that calculates the xylem structure that maximizes carbon-use efficiency while simultaneously taking into account pit structure in increasing the resistance to water transport and constricting the spreading of embolisms. The optimal xylem structure predicted by the model was found to correspond well to the generally observed trends: xylem conduits grew in size from the apex towards the base while simultaneously decreasing in number, and vulnerability to cavitation increased with conduit size. These trends were caused primarily by the axial water potential gradient in the xylem. The pits have to be less porous near the apex where water potential is lower to restrict the spreading of embolisms, while whole-plant carbon-use efficiency demands that conduit size decreases and conduit number increases simultaneously. The model predictions remained qualitatively the same regardless of the exact optimality criterion used for defining carbon-use efficiency.     

Norway spruce(Picea abies) laccases:Characterization of a laccase in a lignin-forming tissue culture

Secondarily thickened cell walls of water-conducting vessels and tracheids and support-giving sclerenchyma cells contain lignin that makes the cell wal s water impermeable and strong. To what extent laccases and peroxidases contribute to lignin biosynthesis in muro is under active evaluation. We performed an in silico study of Norway spruce(Picea abies(L.)Karst.) laccases utilizing available genomic data. As many as 292 laccase encoding sequences(genes, gene fragments, and pseudogenes) were detected in the spruce genome. Out of the 112 genes annotated as laccases, 79 are expressed at some level. We isolated five full-length laccase c DNAs from developing xylem and an extracellular lignin-forming cell culture of spruce. In addition, we purified and biochemically characterized one culture medium laccase from the lignin-forming cell culture.This laccase has an acidic p H optimum(p H 3.8–4.2) for coniferyl alcohol oxidation. It has a high af finity to coniferyl alcohol with an apparent K m value of 3.5 m M; however, the laccase has a lowe catalytic ef ficiency(V max/K m) for coniferyl alcohol oxidation compared with some purified culture medium peroxidases. The properties are discussed in the context of the information already known about laccases/coniferyl alcohol oxidases o coniferous plants.