Targeted transcriptomic and metabolic profiling reveals temporal bottlenecks in the maize carotenoid pathway that may be addressed by multigene engineering

Carotenoids are a diverse group of tetraterpenoid pigments found in plants, fungi, bacteria and some animals. They play vital roles in plants and provide important health benefits to mammals, including humans. We previously reported the creation of a diverse population of transgenic maize plants expressing various carotenogenic gene combinations and exhibiting distinct metabolic phenotypes. Here we performed an in‐depth targeted mRNA and metabolomic analysis of the pathway to characterize the specific impact of five carotenogenic transgenes and their interactions with 12 endogenous genes in four transgenic lines representing distinct genotypes and phenotypes. We reconstructed the temporal profile of the carotenoid pathway during endosperm development at the mRNA and metabolic levels (for total and individual carotenoids), and investigated the impact of transgene expression on the endogenous pathway. These studies enabled us to investigate the extent of any interactions between the introduced transgenic and native partial carotenoid pathways during maize endosperm development. Importantly, we developed a theoretical model that explains these interactions, and our results suggest genetic intervention points that may allow the maize endosperm carotenoid pathway to be engineered in a more effective and predictable manner.     

Uncoupling the chemical steps of telomere resolution by ResT

ResT is the telomere resolvase of the spirochete Borrelia burgdorferi, the causative agent of Lyme disease. ResT is an essential cellular function that processes replication intermediates to produce linear replicons terminated by covalently closed hairpin telomeres. ResT generates these hairpin telomeres in a reaction with mechanistic similarities to those catalyzed by type IB topoisomerases and tyrosine recombinases. We report here, that like most of the tyrosine recombinases, ResT requires interprotomer communication, likely in an in-line synapse, to activate reaction chemistry. Unlike the tyrosine recombinases, however, we infer that the cleavage and strand transfer reactions on the two sides of the replicated telomere occur nearly simultaneously. Nonetheless, the chemical steps of the forward and reverse reactions performed by ResT can occur in a non-concerted fashion (i.e. events on the two sides of the replicated telomere can occur independently). We propose that uncoupling of reaction completion on the two sides of the substrate is facilitated by an early commitment to hairpin formation that is imposed by the precleavage action of the hairpin binding module of the ResT active site.     

Structural and electronic features of the ubiquinone and ubiquinol molecules: molecular dynamics and quantum chemical treatments

The coenzyme Q (CoQ) molecule plays a critical role in the biochemical generation of energy in the form of adenosine triphosphate. Various types of CoQ can be classified according to their number of isoprenoid units in the tail. In human beings, CoQ10 is produced and is necessary for the basic functioning of cells. CoQ10 exists in two forms, as ubiquinone (UQ) and as ubiquinol (UQH₂), which have different roles in the body. Molecular dynamics (MD) simulations for the analysis of the effects of solvents on the structure of the UQ molecule are presented. Besides, semi-empirical molecular orbital PM3 calculation is applied to obtain structural and electronic properties of both the UQ and the UQH₂ molecules. According to the MD simulation, the UQ molecule seems to be flexible both in vacuum and in water. On the other hand, the molecule stays more rigid in methanol. PM3 calculations show that both molecules are quite hydrophobic. Furthermore, UQ is chemically more reactive than UQH₂, but the latter is kinetically more stable than the former.     

Rate constants for the first two chemical steps of eumelanogenesis

The kinetics of the initial cyclization and redox exchange reactions involved in the eumelanogenic pathway have been studied previously but because of the difficulty of detecting the intermediate cyclodopa by optical means (because its absorbance is in the same range as dopa which is present in excess in the experimental system) no accurate value for the redox exchange reaction has so far been obtained and there is no available analytical methodology that can be applied to the successive first- and second-order reactions involved. We have synthesized cyclodopa and examined the kinetics of the formation of dopachrome following the pulse radiolytic generation of dopaquinone in its presence. From this direct measurement we determined that the rate constant of the reaction between cyclodopa and dopaquinone is 5.3 x 10(6)/M/s. Employing this value in a computational model of the combined cyclization and redox exchange reactions we calculate that the observed kinetics of dopaquinone decay and dopachrome formation are compatible with a cyclization rate constant of 3.8/s.     

Chemomechanical coupling of the forward and backward steps of single kinesin molecules

The molecular motor kinesin travels processively along a microtubule in a stepwise manner. Here we have studied the chemomechanical coupling of the hydrolysis of ATP to the mechanical work of kinesin by analysing the individual stepwise movements according to the directionality of the movements. Kinesin molecules move primarily in the forward direction and only occasionally in the backward direction. The hydrolysis of a single ATP molecule is coupled to either the forward or the backward movement. This bidirectional movement is well described by a model of Brownian motion assuming an asymmetric potential of activation energy. Thus, the stepwise movement along the microtubule is most probably due to Brownian motion that is biased towards the forward direction by chemical energy stored in ATP molecules.     

Cell responses to single pheromone molecules may reflect the activation kinetics of olfactory receptor molecules

Olfactory receptor cells of the silkmoth Bombyx mori respond to single pheromone molecules with "elementary" electrical events that appear as discrete "bumps" a few milliseconds in duration, or bursts of bumps. As revealed by simulation, one bump may result from a series of random openings of one or several ion channels, producing an average inward membrane current of 1.5 pA. The distributions of durations of bumps and of gaps between bumps in a burst can be fitted by single exponentials with time constants of 10.2 ms and 40.5 ms, respectively. The distribution of burst durations is a sum of two exponentials; the number of bumps per burst obeyed a geometric distribution (mean 3.2 bumps per burst). Accordingly the elementary events could reflect transitions among three states of the pheromone receptor molecule: the vacant receptor (state 1), the pheromone-receptor complex (state 2), and the activated complex (state 3). The calculated rate constants of the transitions between states are k(21)=7.7 s(-1), k(23)=16.8 s(-1), and k(32)=98 s(-1).     

Computational constraints that may have favoured the lamination of sensory cortex

At the transition from early reptilian ancestors to primordial mammals, the areas of sensory cortex that process topographic modalities acquire the laminar structure of isocortex. A prominent step in lamination is granulation, whereby the formerly unique principal layer of pyramidal cells is split by the insertion of a new layer of excitatory, but intrinsic, granule cells, layer IV. I consider the hypothesis that granulation, and the differentiation between supra- and infra-granular pyramidal layers, may be advantageous to support fine topography in their sensory maps. Fine topography implies a generic distinction between where information, explicitly mapped on the cortical sheet, and what information, represented in a distributed fashion as a distinct firing pattern across neurons. These patterns can be stored on recurrent collaterals in the cortex, and such memory can help substantially in the analysis of current sensory input. The simulation of a simplified network model demonstrates that a non-laminated patch of cortex must compromise between transmitting where information or retrieving what information. The simulation of a modified model including differentiation of a granular layer shows a modest but significant quantitative advantage, expressed as a less severe trade-off between what and where. The further connectivity differentiation between infra-granular and supra-granular pyramidal layers is shown to match the mix of what and where information optimal for their respective target structures.     

Separation of transport and metabolic steps in the uptake of succinate byKluyveromyces fragilis

Characteristics of succinate uptake were determined in glucose-, glycerol-, and succinate-grown cells of the succinate-utilizingKluyveromyces fragilis. Glucose represses the uptake when present during growth and inhibits it when present during uptake. Absence of glucose or presence of succinate or glycerol derepresses the uptake. The transport and subsequent metabolic steps can be efficiently separated by inhibiting the latter with carboxin.     

Trophozoites of Giardia lamblia may have a Golgi-like structure

Trophozoites of the primitive protozoan Giardia lamblia have been considered as cells which do not present the Golgi complex. Using C(6)-NBD ceramide, which has been shown to label the Golgi complex of mammalian cells, labelling of the perinuclear region of G. lamblia was observed by confocal laser scanning microscopy. Transmission electron microscopy of thin sections and of replicas of freeze-fractured cells revealed the presence of concentric perinuclear membranes resembling the Golgi complex.     

Small molecules and chemical tools at the interface

As our understanding of the molecular mechanisms driving complex biological processes grows, the role of chemical biology will continue to expand. The 2008 American Society for Biochemistry and Molecular Biology meeting showcased recent progress in the field of chemical biology while simultaneously pointing toward the future of research at the interface between chemistry and biology.     

Nucleotide synthetase ribozymes may have emerged first in the RNA world

Though the "RNA world" hypothesis has gained a central role in ideas concerning the origin of life, the scenario concerning its emergence remains uncertain. It has been speculated that the first scene may have been the emergence of a template-dependent RNA synthetase ribozyme, which catalyzed its own replication: thus, "RNA replicase." However, the speculation remains uncertain, primarily because of the large sequence length requirement of such a replicase and the lack of a convincing mechanism to ensure its self-favoring features. Instead, we propose a nucleotide synthetase ribozyme as an alternative candidate, especially considering recent experimental evidence suggesting the possibility of effective nonenzymatic template-directed synthesis of RNA. A computer simulation was conducted to support our proposal. The conditions for the emergence of the nucleotide synthetase ribozyme are discussed, based on dynamic analysis on a computer. We suggest the template-dependent RNA synthetase ribozyme emerged later, perhaps after the emergence of protocells.     

Environmental factors which may have led to the appearance of colour vision

It is hypothesized that colour vision and opponent processing of colour signals in the visual system evolved as a means of overcoming the extremely unfavourable lighting conditions in the natural environment of early vertebrates. The significant flicker of illumination inherent in the shallow-water environment complicated the visual process in the achromatic case, in particular preventing early detection of enemies. The presence of two spectral classes of photoreceptors and opponent interaction of their signals at a subsequent retinal level allowed elimination of the flicker from the retinal image. This new visual function provided certain advantages concerning reaction times and favoured survival. This assumption explains why the building blocks for colour vision arose so early, i.e. just after the active predatory lifestyle was mastered. The principal functions of colour vision inherent in extant animals required a more complex neural machinery for colour processing and evolved later as the result of a change in visual function favouring colour vision.     

Site-specific chemical labeling of long RNA molecules

Site-specific labeling of RNA molecules is a valuable tool for studying their structure and function. Here, we describe a new site-specific RNA labeling method, which utilizes a DNA-templated chemical reaction to attach a label at a specific internal nucleotide in an RNA molecule. The method is nonenzymatic and based on the formation of a four-way junction, where a donor strand is chemically coupled to an acceptor strand at a specific position via an activated chemical group. A disulfide bond in the linker is subsequently cleaved under mild conditions leaving a thiol group attached to the acceptor-RNA strand. The site-specific thiol-modified target RNA can then be chemically labeled with an optional group, here demonstrated by coupling of a maleimide-functionalized fluorophore. The method is rapid and allows site specific labeling of both in vitro and in vivo synthesized RNA with a broad range of functional groups.     

Gentamicin may have an influence on the effectiveness of peritoneal dialysis]

An effect of gentamicin on urea and uric acid transfer through peritoneum was studied in rabbits in vitro. It was found that gentamicin decreases urea transfer by approximately 30%. Its effect on uric acid transfer was transient so as mean values of this parameter did not differ statistically before and after antibiotic administration. It may be assumed that gentamicin decreases permeability of peritoneum for certain low molecules of endogenic origin. Such an effect may prove unfavourable when peritoneal dialysis efficiency is concerned.     

Structural and chemical features of the invertebrate otoliths

In the course of evolution of the invertebrate gravity receptor endogenous intracellular mode of the otoliths formation (Coelenterata, Ctenophora) is changed for endogenous extracellular mode (Mollusca, Arthropoda). The intracellular otolith is represented by cristalline concretions whereas extracellular one may consists of many separate crystallic otoconia or one large spherolith. As a rule in all cases qualitative mineral composition is similar. Calcium is the main component of the otolith in invertebrates (as vertebrates) though magnesium, sodium and potassium also take place in the otolith formation.     

Streptozotocin-induced diabetes modulates the metabolic activation of chemical carcinogens

The effect of chemically-induced diabetes on the hepatic microsomal mixed-function oxidase system and the activation of chemical carcinogens was investigated in animals treated with streptozotocin (STZ). In order to distinguish between the effects of the diabetogenic chemical per se and that of the diabetic state, groups of STZ-treated animals received either nicotinamide simultaneously with STZ to prevent the onset of diabetes, or daily treatment with insulin in order to reverse the effects of diabetes. STZ-treated animals exhibited higher pentoxyresorufin O-dealkylase, ethoxy-resorufin O-deethylase, ethoxycoumarin O-deethylase, aniline p-hydroxylase and NADPH-cytochrome c reductase activities; similarly, increases were seen in cytochrome P-450 and b5 levels. All of these effects were prevented by nicotinamide and, at least partly, antagonised by insulin therapy. Treatment of animals with STZ markedly increased the activation, by liver microsomes in vitro, of Trp-P-1 and Trp-P-2 to mutagens, the effect being totally preventable by nicotinamide and successfully antagonised with insulin therapy. The diabetic animals were similarly more efficient in activating MeIQ but the effect was not preventable by nicotinamide or reversed by insulin. In contrast no changes were seen in the activation of IQ and only a modest increase in the case of MeIQx. It is concluded that diabetes may modulate the metabolic activation of some chemical carcinogens, presumably by changing the ratio of the various cytochrome P-450 isoenzymes.