Engineering key components in a synthetic eukaryotic signal transduction pathway

Signal transduction underlies how living organisms detect and respond to stimuli. A goal of synthetic biology is to rewire natural signal transduction systems. Bacteria, yeast, and plants sense environmental aspects through conserved histidine kinase (HK) signal transduction systems. HK protein components are typically comprised of multiple, relatively modular, and conserved domains. Phosphate transfer between these components may exhibit considerable cross talk between the otherwise apparently linear pathways, thereby establishing networks that integrate multiple signals. We show that sequence conservation and cross talk can extend across kingdoms and can be exploited to produce a synthetic plant signal transduction system. In response to HK cross talk, heterologously expressed bacterial response regulators, PhoB and OmpR, translocate to the nucleus on HK activation. Using this discovery, combined with modification of PhoB (PhoB‐VP64), we produced a key component of a eukaryotic synthetic signal transduction pathway. In response to exogenous cytokinin, PhoB‐VP64 translocates to the nucleus, binds a synthetic PlantPho promoter, and activates gene expression. These results show that conserved‐signaling components can be used across kingdoms and adapted to produce synthetic eukaryotic signal transduction pathways.     

Dextrose and Sorbitol as Diluents for Continuous Intravenous Heparin Infusion

A comparison of 5% dextrose and 5% sorbitol as diluents for heparin given by continuous intravenous infusion indicated that neither impaired the potency of the heparin. Previous suggestions that heparin becomes unstable in dextrose solution have not been confirmed.     

RNA interference: past, present and future

RNA interference (RNAi) is the sequence-specific gene silencing induced by double-stranded RNA. RNAi is mediated by 21-23 nucleotide small interfering RNAs (siRNAs) which are produced from long double-stranded RNAs by RNAse II-like enzyme Dicer. The resulting siRNAs are incorporated into a RNA-induced silencing complex (RISC) that targets and cleaves mRNA complementary to the siRNAs. Since its inception in 1998, RNAi has been demonstrated in organisms ranging from trypanosomes to nematodes to vertebrates. Potential uses already in progress include the examination of specific gene function in living systems, the development of anti-viral and anti-cancer therapies, and genome-wide screens. In this review, we discuss the landmark discoveries that established the contextual framework leading up to our current understanding of RNAi. We also provide an overview of current developments and future applications.     

Ontogenetic changes in Mammalian feeding: insights from electromyographic data

All infant mammals make a transition from suckling milk to eating solid foods. Yet, the neuromuscular implications of the transition from a liquid-only diet to solid foods are unknown even though the transport and swallowing of liquids is different from that of solids. We used legacy electromyography (EMG) data to test hypotheses concerning the changes in motor pattern and neuromuscular control that occur during the transition from an all-liquid diet to consumption of solid food in a porcine model. EMG signals were recorded from five oropharyngeal muscles in pigs at three developmental stages (infants, juveniles, and adults) feeding on milk, on food of an intermediate consistency (porridge), and on dry chow (juveniles and adults only). We measured cycle frequency and its variation in "transport cycles" and "swallow cycles". In the swallow cycles, a measure of variation of the EMG signal was also calculated. Variation in cycle frequency for transport and swallow cycles was lowest in adults, as predicted, suggesting that maturation of feeding mechanisms occurs as animals reach adulthood. Infants had lower variation in transport cycle frequency than did juveniles drinking milk, which may be due to the greater efficiency of the infant's tight oral seal against the teat during suckling, compared to a juvenile drinking from a bowl where a tight seal is not possible. Within juveniles, variation in both transport and swallow cycle frequencies was directly related to food consistency, with the highest variation occurring when drinking milk and the lowest when feeding on solid food. There was no difference in the variation of the EMG activity between intact infants and juveniles swallowing milk, although when the latter swallow porridge the EMG signals were less variable than for milk. These results suggest that consistency of food is a highly significant determinant of the variation in motor pattern, particularly in newly weaned animals.     

How dogs lap: ingestion and intraoral transport in Canis familiaris

It has recently been suggested that the mechanism for lifting liquid from a bowl into the oral cavity during lapping is fundamentally different in cats and dogs: cats use adhesion of liquid to the tongue tip while dogs 'scoop' with their backwardly curled tongue. High-speed light videos and X-ray videos show that on the contrary, both cats and dogs use the mechanism of adhesion. Liquid is transported through the oral cavity to the oesophagus, against gravity, on the surface of the tongue as it is drawn upwards, then a tight contact between the tongue surface and palatal rugae traps liquid and prevents its falling out as the tongue is protruded. At least three cycles are needed for intraoral transport of liquid in the dog.     

Natural and induced cadmium-accumulation in poplar and willow: Implications for phytoremediation

Potentially poplars and willows may be used for the in situ decontamination of soils polluted with Cd, such as pasturelands fertilised with Cd-rich superphosphate fertiliser. Poplar (Kawa and Argyle) and willow (Tangoio) clones were grown in soils containing a range (0.6–60.6 μg g⁻¹ dry soil) of Cd concentrations. The willow clone accumulated significantly more Cd (9–167 μg g⁻¹ dry matter) than the two poplar clones (6–75 μg g⁻¹), which themselves were not significantly different. Poplar trees (Beaupré) sampled in situ from a contaminated site near the town of Auby, Northern France, were also found to accumulate significant quantities (up to 209 μg g⁻¹) of Cd. The addition of chelating agents (0.5 and 2 g kg⁻¹ EDTA, 0.5 g kg⁻¹ DTPA and 0.5 g kg⁻¹NTA) to poplar (Kawa) clones caused a temporary increase in uptake of Cd. However, two of the chelating agents (2 g kg⁻¹ EDTA and 0.5 g kg⁻¹ NTA) also resulted in a significant reduction in growth, as well as abscission of leaves. If the results obtained in these pot experiments can be realised in the field, then a single crop of willows could remove over 100 years worth of fertiliser-induced Cd contamination from pasturelands. This revised version was published online in June 2006 with corrections to the Cover Date.     

Evolution of developmentally regulated genome rearrangements in eukaryotes

Developmentally regulated genome rearrangements (DRGR)--processes that alter genomes either in specific cells or during specific life cycle stages--are widespread throughout eukaryotes. This contrasts with the view that genome structure and content remain essentially constant throughout an organism's life cycle. Here we review three categories of developmentally regulated genome processing in eukaryotes: genome-wide rearrangements, targeted rearrangements, and a special case of amplification of ribosomal DNA genes. Mapping these types of DRGR onto eukaryotic phylogeny indicates that each type of processing is found in multiple independent lineages. We propose that such genome rearrangements were present within the last common ancestor of extant eukaryotes, and that future research will yield evidence of homologous epigenetic mechanisms underlying genome processing among diverse eukaryotes.     

A conserved N-capping motif contributes significantly to the stabilization and dynamics of the C-terminal region of class Alpha glutathione S-transferases

Helix 9, the major structural element in the C-terminal region of class Alpha glutathione transferases, forms part of the active site of these enzymes where its dynamic properties modulate both catalytic and ligandin functions. A conserved aspartic acid N-capping motif for helix 9 was identified by sequence alignments of the C-terminal regions of class Alpha glutathione S-transferases (GSTs) and an analysis by the helix-coil algorithm AGADIR. The contribution of the N-capping motif to the stability and dynamics of the region was investigated by replacing the N-cap residue Asp-209 with a glycine in human glutathione S-transferase A1-1 (hGST A1-1) and in a peptide corresponding to its C-terminal region. Far-UV circular dichroism and AGADIR analyses indicate that, in the absence of tertiary interactions, the wild-type peptide displays a low intrinsic tendency to form a helix and that this tendency is reduced significantly by the Asp-to-Gly mutation. Disruption of the N-capping motif of helix 9 in hGST A1-1 alters the conformational dynamics of the C-terminal region and, consequently, the features of the H-site to which hydrophobic substrates (e.g. 1-chloro-2,4-dinitrobenzene (CDNB)) and nonsubstrates (e.g. 8-anilino-1-naphthalene sulfonate (ANS)) bind. Isothermal calorimetric and fluorescence data for complex formation between ANS and protein suggest that the D209G-induced perturbation in the C-terminal region prevents normal ligand-induced localization of the region at the active site, resulting in a less hydrophobic and more solvent-exposed H-site. Therefore, the catalytic efficiency of the enzyme with CDNB is diminished due to a lowered affinity for the electrophilic substrate and a lower stabilization of the transition state.