A combined approach to improving large-scale production of tobacco etch virus protease

Tobacco etch virus NIa proteinase (TEV protease) is an important tool for the removal of fusion tags from recombinant proteins. Production of TEV protease in Escherichia coli has been hampered by insolubility and addressed by many different strategies. However, the best previous results and newer approaches for protein expression have not been combined to test whether further improvements are possible. Here, we use a quantitative, high-throughput assay for TEV protease activity in cell lysates to evaluate the efficacy of combining several previous modifications with new expression hosts and induction methods. Small-scale screening, purification and mass spectral analysis showed that TEV protease with a C-terminal poly-Arg tag was proteolysed in the cell to remove four of the five arginine residues. The truncated form was active and soluble but in contrast, the tagged version was also active but considerably less soluble. An engineered TEV protease lacking the C-terminal residues 238-242 was then used for further expression optimization. From this work, expression of TEV protease at high levels and with high solubility was obtained by using auto-induction medium at 37 degrees C. In combination with the expression work, an automated two-step purification protocol was developed that yielded His-tagged TEV protease with >99% purity, high catalytic activity and purified yields of approximately 400 mg/L of expression culture (approximately 15 mg pure TEV protease per gram of E. coli cell paste). Methods for producing glutathione-S-transferase-tagged TEV with similar yields (approximately 12 mg pure protease fusion per gram of E. coli cell paste) are also reported.     

A photographic approach to the possible mechanism of retrogradation of sweet potato starch

Although the subject of starch retrogradation has been studied for about 20 years, the mechanism of starch retrogradation seems not yet to be completely established. In this paper, the possible retrogradation mechanism of sweet potato starch was postulated from four optical micrographs at the stages of melting of the starch granules, autoclaving treatment and aging. The possible process of retrogradation consists of three stages. Firstly, starch granules was swelled and melted with loss of X-ray crystallinity and formation of both crystalline and amorphous lamellae; secondly, in crystalline lamellae, amylopectin began to form nucleation when they were autoclaved; finally, the nucleus grew up to great rod-like crystals as the result of congregating of amylose on plates which were composed of and prolongated by amylopectin.     

A note on a possible mathematical approach to the theory of individual freedom

As suggested in previous publications, freedom may be defined quantitatively as a restriction upon the choice of a number of activities. If the choice is determined by maximizing the satisfaction function, it is suggested that freedom may be defined in terms of the satisfaction function. If an individual is isolated and no physical restrictions limit his choice of activities, he is free to choose any activity in an amount which maximizes his satisfaction. This isolated state may be considered therefore as that of maximum freedom. If the individual interacts with another, he will choose different amounts of his object of satisfaction depending on whether he behaves egoistically or altruistically. But in either case the value chosen will not maximize his satisfaction function considered alone. A simple analytical expression is suggested as a measure of freedom in this case, and some problems which arise from this suggestion are mentioned.     

A symmetry approach to electrocardiogram analysis

A qualitative characteristics of a symmetry of correlations in ECG parameters has been studied in experiments on rats with the adaptation syndrome and hypertonic patients (I-II stages). A geometrical model of cardiac cycle shaped as a rectangular triangle with temporal and amplitude ECG parameters as catheti. A total correlation between the ECG parameters is given: QT--QRS/QT--PQ.RR--PQ/RR--QRS = R--P/R--T = 1,309. In healthy individuals, the deviation from the ideal proportion constitutes less than 5%, and in patients with cardiovascular diseases, the deviations are substantial.     

A vector projection approach to predicting HIV protease cleavage sites in proteins

A vector projection method is proposed to predict the cleavability of oligopeptides by extended-specificity site proteases. For an enzyme with eight specificity subsites the substrate octapeptide can be uniquely expressed as a vector in an 8-dimensional space, whose eight bases correspond to the amino acids at the eight subsites, P₁, P_1′, P_2′, P_3′ and P_4′, respectively. The component of such a characteristic vector on each of the eight bases is defined as the frequency of an amino acid occurring at a given site. These frequencies were derived from a set of octapeptides known to be cleaved by HIV protease. The cleavability of an octapeptide can then be estimated from the projection of its characteristic vector on an idealized, optimally cleavable vector. The high ratio of correct prediction vs. total prediction for the data in both the training and the testing sets indicates that the new method is self-consistent and efficient. It provides a rapid and accurate algorithm for analyzing the specificity of any multisubsite enzyme for which there is no coupling between subsites. In particular, it is useful for predicting the cleavability of an oligopeptide by either HIV-1 or HIV-2 protease, and hence offers a supplementary means for finding effective inhibitors of HIV protease as potential drugs against AIDS. © Wiley-Liss, Inc.     

A possible approach to the investigation of reacting sites of ozone in rat lung

As edematogenic agents, thiocarbamides and ozone share several pharmacological and toxicological features in common. In view of these striking similarities, an attempt was made to find out whether or not thiocarbamides and ozone interact with the same cellular constituents of the lungs. This was accomplished by incubating first, a constant amount of ¹⁴C-thiourea with lung slices of O₃-tolerant and control rats followed by subcellular fractionation and radio-activity determination. The results of this study reveal a substantial and significant reduction in binding of ¹⁴C-thiourea to all sub-cellular fractions of lungs of O₃-tolerant rats as compared to the rats exposed to ambient air. The data suggest that repeated exposure to a progressively increasing concentration of ozone inhibits ¹⁴C-thiourea binding in the lung by reducing binding sites by directly destroying binding sites or via an effect on cell population.     

A new approach to possible substrate binding mechanisms for nitrile hydratase

We combined normal mode analysis (NMA) with cavity calculations as a method to get more insight into static crystal structures. We used nitrile hydratase (NHase) as a case study, and the crystal structure of a complex of Pseudonocardia thermophila NHase (1UGP) with n-butyric acid was chosen as a reference structure. The reference structure was compared with the other available NHase crystal structures. Cavity calculations of the static structures showed the entrances to the active site and also a possible function of the N-terminal in the substrate selection of the Co-type NHase. When NMA was combined with cavity calculations, a closing-opening passage was observed. Analysis of low frequency modes combined with cavity calculations led us to propose "breathing" and "flip-flop" mechanisms which might be a key part of the substrate binding mechanism.     

A semi-quantitative approach to GMO risk-benefit analysis

In many countries there are increasing calls for the benefits of genetically modified organisms (GMOs) to be considered as well as the risks, and for a risk-benefit analysis to form an integral part of GMO regulatory frameworks. This trend represents a shift away from the strict emphasis on risks, which is encapsulated in the Precautionary Principle that forms the basis for the Cartagena Protocol on Biosafety, and which is reflected in the national legislation of many countries. The introduction of risk-benefit analysis of GMOs would be facilitated if clear methodologies were available to support the analysis. Up to now, methodologies for risk-benefit analysis that would be applicable to the introduction of GMOs have not been well defined. This paper describes a relatively simple semi-quantitative methodology that could be easily applied as a decision support tool, giving particular consideration to the needs of regulators in developing countries where there are limited resources and experience. The application of the methodology is demonstrated using the release of an insect resistant maize variety in South Africa as a case study. The applicability of the method in the South African regulatory system is also discussed, as an example of what might be involved in introducing changes into an existing regulatory process.     

A multi-dimensional scaling approach to shape analysis

We propose an alternative to Kendall's shape space for reflectionshapes of configurations in with k labelled vertices, where reflection shape consistsof all the geometric information that is invariant under compositionsof similarity and reflection transformations. The proposed approachembeds the space of such shapes into the space of (k – 1) x (k – 1) real symmetricpositive semidefinite matrices, which is the closure of an opensubset of a Euclidean space, and defines mean shape as the naturalprojection of Euclidean means in on to the embedded copy of the shape space. This approachhas strong connections with multi-dimensional scaling, and themean shape so defined gives good approximations to other commonlyused definitions of mean shape. We also use standard perturbationarguments for eigenvalues and eigenvectors to obtain a centrallimit theorem which then enables the application of standardstatistical techniques to shape analysis in two or more dimensions.     

A DSRPCL-SVM approach to informative gene analysis

Microarray data based tumor diagnosis is a very interesting topic in bioinformatics. One of the key problems is the discovery and analysis of informative genes of a tumor. Although there are many elaborate approaches to this problem, it is still difficult to select a reasonable set of informative genes for tumor diagnosis only with microarray data. In this paper, we classify the genes expressed through microarray data into a number of clusters via the distance sensitive rival penalized competitive learning （DSRPCL） algorithm and then detect the informative gene cluster or set with the help of support vector machine （SVM）. Moreover, the critical or powerful informative genes can be found through further classifications and detections on the obtained informative gene clusters. It is well demonstrated by experiments on the colon, leukemia, and breast cancer datasets that our proposed DSRPCL-SVM approach leads to a reasonable selection of informative genes for tumor diagnosis.     

A stochastic approach to phyllotactic patterns analysis

A statistical method is presented to characterize the degree of order in phyllotactic systems. We developed equations allowing the theoretical estimation of the number of leaves regularly distributed (spiral or verticillate) in a partially random phyllotactic system. The equations are simple and accurate enough to make quantitative predictions concerning the organization of different phyllotactic patterns (verticillate, distichous, spiral and random). This method can bring out patterns that are not visible a priori on a planar representation of the shoot apex. As a case study, the method was applied to the quantitative analysis of the sho mutants recently produced by Itoh et al. [2000. SHOOT ORGANIZATION genes regulate shoot apical meristem organization and the pattern of leaf primordium initiation in Rice. Plant Cell 12, 2161-2174]. By using our method, it was possible to predict the number of leaves distributed in distichous or random patterns on these phyllotactic mutants.     

A comparative neuropsychological approach to the analysis of hippocampal functions

Formation of conditioned switching-over of heterogeneous instrumental reflexes in dogs was more successful than in rats, which testifies about significant differences of the functional organization of analytical-synthetic brain activity in rodents and predatory animals. Experiments with lesion of the hippocampus (in rats) and the records of its electrical activity (in dogs) allowed to conclude that the hippocampus of the both types of animals belongs to the system of structures, participating in the formation of conditioned switching-over. The data, obtained in the process of elaboration of the switching-over and probabilistic reinforcement of the alimentary conditioned stimulus, testify that providing for the reactions to signals with low probability of reinforcement in different species of animals is one of the functions of the hippocampus.