Optimization of bovine serum albumin sorption and recovery by hydrogels

Aqueous two-phase systems are composed of aqueous solutions of either two water-soluble polymers, usually polyethylene glycol (PEG) and dextran (Dx), or a polymer and a salt, usually PEG and phosphate or sulfate. Partitioning of proteins in such systems provides a powerful method for separating and purifying mixtures of biomolecules by extraction. If one of the phase forming polymers is a crosslinked gel, then the solution-controlled gel sorption may be considered as a modification of aqueous two-phase extraction. Since PEG/dextran systems are widely used in aqueous two-phase extraction and dextran gels (Sephadex) are common chromatographic media, we choose a PEG/dextran gel system as a model system in this study. The partitioning behavior of pure bovine serum albumin (BSA) in PEG/dextran gel systems is investigated to see the effects of variations in PEG and NaCl concentrations on the partition coefficient K. By making use of the Box-Wilson experimental design, K is shown to be maximized at 9.8 (%, w/w) PEG and 0.2 M NaCl concentrations, respectively, as 182.     

Computer control of pH and DO in a laboratory fermenter using a neural network technique

In this contribution, the advantages of the artificial neural network approach to the identification and control of a laboratory-scale biochemical reactor are demonstrated. It is very important to be able to maintain the levels of two process variables, pH and dissolved oxygen (DO) concentration, over the course of fermentation in biosystems control. A PC-supported, fully automated, multi-task control system has been designed and built by the authors. Forward and inverse neural process models are used to identify and control both the pH and the DO concentration in a fermenter containing a Saccharomyces cerevisiae based-culture. The models are trained off-line, using a modified back-propagation algorithm based on conjugate gradients. The inverse neural controller is augmented by a new adaptive term that results in a system with robust performance. Experimental results have confirmed that the regulatory and tracking performances of the control system proposed are good.     

New targets of Arabidopsis thioredoxins revealed by proteomic analysis

Proteomics was used to search for putative thioredoxin (TRX) targets in leaves of the model plant, Arabidopsis thaliana. About forty different proteins have been found to be reduced by TRX, after TRX itself has been specifically reduced by its NADPH-dependent reductase. Twenty-one of the identified proteins were already known or recently proposed to be TRX-dependent and nineteen of the proteins were new potential targets. The identified proteins are involved in a wide variety of processes, including the Calvin cycle, metabolism, photosynthesis, folding, defense against oxidative stress and amino acid synthesis. Two proteins from the glycine cleavage complex were also identified as putative TRX targets, and a new role can be postulated in leaves for TRX in defense against herbivores and/or pathogens.     

Heterostyly: speciation within a species

Almost all organisms in nature show nonrandom mating to different degrees. Two extreme results of nonrandom mating are speciation and sexual differentiation. Heterostyly is a form of sexual differentiation considered to have evolved to resolve conflicts between male and female functions of hermaphrodite flowers. Our study examines necessary and sufficient conditions for establishment of heterostyly using a configuration individual-based model. Previous models assume invasion of a mutant phenotype into a population with monomorphic wild phenotype. In contrast, our model demonstrates that heterostyly can be established from a population with continuous phenotypic variation, a proposition that requires simpler assumptions than the previous hypotheses. Results of our simulation show that genetic linkage between stigma and anther heights is essential for establishment of heterostyly. Dominance effects on the genes for stamen or stigma heights are not necessary, but they promote evolution of heterostyly. Probability of evolution of heterostyly also depends on the functional relationship between stigma–anther distance and strength of sexual interference, and the distance and probability of pollen deposition success. Parallelity and difference between speciation and sexual differentiation are also discussed.     

Parallel processing in an identified neural circuit: the Aplysia californica gill-withdrawal response model system

The response of the gill of Aplysia calfornica Cooper to weak to moderate tactile stimulation of the siphon, the gill-withdrawal response or GWR, has been an important model system for work aimed at understanding the relationship between neural plasticity and simple forms of non-associative and associative learning. Interest in the GWR has been based largely on the hypothesis that the response could be explained adequately by parallel monosynaptic reflex arcs between six parietovisceral ganglion (PVG) gill motor neurons (GMNs) and a cluster of sensory neurons termed the LE cluster. This hypothesis, the Kupfermann-Kandel model, made clear, falsifiable predictions that have stimulated experimental work for many years. Here, we review tests of three predictions of the Kupfermann-Kandel model: (1) that the GWR is a simple, reflexive behaviour graded with stimulus intensity; (2) that central nervous system (CNS) pathways are necessary and sufficient for the GWR; and (3) that activity in six identified GMNs is sufficient to account for the GWR. The available data suggest that (1) a variety of action patterns occur in the context of the GWR; (2) the PVG is not necessary and the diffuse peripheral nervous system (PNS) is sufficient to mediate these action patterns; and (3) the role of any individual GMN in the behaviour varies. Both the control of gill-withdrawal responses, and plasticity in these responses, are broadly distributed across both PNS and CNS pathways. The Kupfermann-Kandel model is inconsistent with the available data and therefore stands rejected. There is, no known causal connection or correlation between the observed plasticity at the identified synapses in this system and behavioural changes during non-associative and associative learning paradigms. Critical examination of these well-studied central pathways suggests that they represent a 'wetware' neural network, architecturally similar to the neural network models of the widely used 'Perceptron' and/or 'Back-propagation' type. Such models may offer a more biologically realistic representation of nervous system organisation than has been thought. In this model, the six parallel GMNs of the CNS correspond to a hidden layer within one module of the gill-control system. That is, the gill-control system appears to be organised as a distributed system with several parallel modules, some of which are neural networks in their own right. A new model is presented here which predicts that the six GMNs serve as components of a 'push-pull' gain control system, along with known but largely unidentified inhibitory motor neurons from the PVG. This 'push-pull' gain control system sets the responsiveness of the peripheral gill motor system. Neither causal nor correlational links between specific forms of neural plasticity and behavioural plasticity have been demonstrated in the GWR model system. However, the GWR model system does provide an opportunity to observe and describe directly the physiological and biochemical mechanisms of distributed representation and parallel processing in a largely identifiable 'wetware' neural network.     

Dorsal telencephalon-specific expression of Cre recombinase in PAC transgenic mice

The ability to restrict gene expression or disruption to specific regions of the brain would enhance understanding of the molecular basis for brain development and function. For this purpose, brain region-restricted promoters are essential. Here we report the isolation of a DNA fragment containing the Emx1 gene promoter, which is responsible for dorsal telencephalon-specific expression. The Cre recombinase gene was inserted into a mouse PAC (P1-derived artificial chromosome) Emx1-locus clone (PAC-Emx1#1 clone) and utilized to generate three transgenic mouse lines. In all three lines, especially Tg3, Cre-mediated recombination was highly restricted to Emx1-expressing cell lineages, from embryonic stages to adulthood. Immunohistochemical analyses showed that Cre protein is expressed in the dorsal telencephalon in all three lines in adulthood. Thus, the PAC-Emx1#1 clone contains essentially all regulatory elements necessary for Emx1 gene expression. Our results suggest that Emx1-Cre Tg3 mice and the PAC-Emx1#1 clone constitute powerful tools for dorsal telencephalon-specific gene manipulation.     

Multiple paternity in an aggregate breeding amphibian: the effect of reproductive skew on estimates of male reproductive success

Aggregate, or explosive, breeding is widespread among vertebrates and likely increases the probability of multiple paternity. We assessed paternity in seven field-collected clutches of the explosively breeding spotted salamander (Ambystoma maculatum) using 10 microsatellite loci to determine the frequency of multiple paternity and the number of males contributing to a female's clutch. Using the Minimum Method of allele counts, multiple paternity was evident in 70% of these egg masses. Simple allele counts underestimate the number of contributing males because this method cannot distinguish multiple fathers with common or similar alleles. Therefore, we used computer simulations to estimate from the offspring genotypes the most likely number of contributing fathers given the distributions of allele frequencies in this population. We determined that two to eight males may contribute to A. maculatum clutches; therefore, multiple paternity is a common strategy in this aggregate breeding species. In aggregate mating systems competition for mates can be intense, thus differential reproductive success (reproductive skew) among males contributing to a female's clutch could be a probable outcome. We use our data to evaluate the potential effect of reproductive skew on estimates of the number of contributing males. We simulated varying scenarios of differential male reproductive success, ranging from equal contribution to high reproductive skew among contributing sires in multiply sired clutches. Our data suggest that even intermediate levels of reproductive skew decrease confidence substantially in estimates of the number of contributing sires when parental genotypes are unknown.     

Effective size of two feral domestic cat populations (Felis catus L): effect of the mating system

A variety of behavioural traits have substantial effects on the gene dynamics and genetic structure of local populations. The mating system is a plastic trait that varies with environmental conditions in the domestic cat (Felis catus) allowing an intraspecific comparison of the impact of this feature on genetic characteristics of the population. To assess the potential effect of the heterogenity of males' contribution to the next generation on variance effective size, we applied the ecological approach of Nunney & Elam (1994) based upon a demographic and behavioural study, and the genetic 'temporal methods' of Waples (1989) and Berthier et al. (2002) using microsatellite markers. The two cat populations studied were nearly closed, similar in size and survival parameters, but differed in their mating system. Immigration appeared extremely restricted in both cases due to environmental and social constraints. As expected, the ratio of effective size to census number (Ne/N) was higher in the promiscuous cat population (harmonic mean = 42%) than in the polygynous one (33%), when Ne was calculated from the ecological method. Only the genetic results based on Waples' estimator were consistent with the ecological results, but failed to evidence an effect of the mating system. Results based on the estimation of Berthier et al. (2002) were extremely variable, with Ne sometimes exceeding census size. Such low reliability in the genetic results should retain attention for conservation purposes.     

Characterization of a novel toxin-antitoxin module, VapBC, encoded by Leptospira interrogans chromosome

Comparative genomic analysis of the coding sequences (CDSs) of Leptospira interrogans revealed a pair of closely linked genes homologous to the vapBC loci of many other bacteria with respect to both deduced amino acid sequencesand operon organizations. Expression of single vapC gene in Escherichia coli resulted in inhibition of bacterial growth,whereas co-expression of vapBC restored the growth effectively. This phenotype is typical for three other character-ized toxin-antitoxin systems of bacteria, i.e., mazEF[1], reIBE[2] and chplK[3]. The VapC proteins of bacteria and a thermophilic archeae, Solfolobus tokodaii, form a structurally distinguished group of toxin different from the other known toxins of bacteria. Phylogenetic analysis of both toxins and antitoxins of all categories indicated that althought oxins were evolved from divergent sources and may or may not follow their speciation paths (as indicated by their 16s RNA seouences）, co-evolution with their antitoxins was obvious.