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The Catharanthus (or Vinca )alkaloids comprise a group of about 130 terpenoid indole alkaloids. Vinblastine is now marketed for more than 40 years as an anticancer drug and became a true lead compound for drug development.Due to the pharmaceutical importance and the low content in the plant of vinblastine and the related alkaloid vincristine,Catharanthus roseus became one of the best-studied medicinal plants. Consequently it developed as a model system for biotechnological studies on plant secondary metabolism. The aim of this review is to acquaint a broader audience with the recent progress in this research and with its exciting perspectives.The pharmacognostical aspects of the Catharanthus alkaloids cover botanical (including some historical),phytochemical and analytical data.An up-to-date view on the biosynthesis of the alkaloids is given.The pharmacological aspects of these alkaloids and their semi-synthetic derivatives are only discussed briefly. The biotechnological part focuses on alternative production systems for these alkaloids,for example by in vitro culture of C.roseus cells.Subsequently it will be discussed to what extent the alkaloid biosynthetic pathway can be manipulated genetically (\"metabolic engineering \"),aiming at higher production levels of the alkaloids.Another approach is to produce the alkaloids (or their precursors)in other organisms such as yeast. Despite the availability of only a limited number of biosynthetic genes,the research on C.roseus has already led to a broad scientific spin-off.It is clear that many interesting results can be expected when more genes become available.

Abstract Kluyveromyces lactis is both scientifically and biotechnologically one of the most important non-Saccharomyces yeasts. Its biotechnological significance builds on its history of safe use in the food industry and its well-known ability to produce enzymes like lactase and bovine chymosin on an industrial scale. In this article, we review the various strains, genetic techniques and molecular tools currently available for the use of K. lactis as a host for protein expression. Additionally, we present data illustrating the recent use of proteomics studies to identify cellular bottlenecks that impede heterologous protein expression.

A proteomic approach is undertaken aiming at the identification of novel proteins involved in the alkaloid biosynthesis of Catharanthus roseus. The C. roseus cell suspension culture A11 accumulates the terpenoid indole alkaloids strictosidine, ajmalicine and vindolinine. Cells were grown for 21 days, and alkaloid accumulation was monitored during this period. After a rapid increase between day 3 and day 6, the alkaloid content reached a maximum on day 16. Systematic analysis of the proteome was performed by two-dimensional polyacrylamide gel electrophoresis. After day 3, the proteome started to change with an increasing number of protein spots. On day 13, the proteome changed back to roughly the same as at the start of the growth cycle. 88 protein spots were selected for identification by mass spectrometry (MALDI-MS/MS). Of these, 58 were identified, including two isoforms of strictosidine synthase (EC 4.3.3.2), which catalyzes the formation of strictosidine in the alkaloid biosynthesis; tryptophan synthase (EC 4.1.1.28), which is needed for the supply of the alkaloid precursor tryptamine; 12-oxophytodienoate reductase, which is indirectly involved in the alkaloid biosynthesis as it catalyzes the last step in the biosynthesis of the regulator jasmonic acid. Unique sequences were found, which may also relate to unidentified biosynthetic proteins.

Accurate label-free methods or assays to obtain the initial reaction rates have significant importance in fundamental studies of enzymes and in application-oriented high throughput screening of enzyme activity. Here we introduce a label-free approach for obtaining initial rates of enzyme activity from heat measurements, which we name initial rate calorimetry (IrCal). This approach is based on our new finding that the data recorded by isothermal titration calorimetry for the early stages of a reaction, which have been widely ignored, are correlated to the initial rates. Application of the IrCal approach to various enzymes led to accurate enzyme kinetics parameters as compared to spectroscopic methods and enabled enzyme kinetic studies with natural substrate, e.g. proteases with protein substrates. Because heat is a label-free property of almost all reactions, the IrCal approach holds promise in fundamental studies of various enzymes and in use of calorimetry for high throughput screening of enzyme activity.

A nested polymerase chain reaction (PCR) was used to detect and identify mycoplasma contaminants in viral stocks. The results of the PCR assay proved to be a sensitive and accurate indicator of the true status of the stock tested. Those samples positive by agar culture or Hoechst stain were also positive by PCR. Those samples that were inconclusive by Hoechst stain (10.05%) could be clearly determined to be mycoplasma positive or negative by PCR. The PCR assay also detected those fastidious species of mycoplasma that gave false negative results by the direct culture method. In many respects the PCR-based mycoplasma detection method described is superior to the agar culture and Hoechst staining detection methods. In this study, the PCR assay detected substantially more mycoplasma-positive viral stocks than did the agar culture assay. Due to its speed, sensitivity, and reliability, the PCR assay is of particular value in monitoring the process of removing mycoplasma from contaminated stocks. Furthermore, the PCR amplification products can be analyzed by restriction analysis to rapidly identify the species of the mycoplasma contaminating the stock tested.

With the establishment of the European Security and Defence Policy (ESDP) in 1999, the Common Foreign and Security Policy (CFSP) acquired an operational aspect that had been absent up until now. Under the framework of ESDP, the European Union (EU) has launched almost 25 civilian and military crisis management operations across the world between January 2003 and August 2010, with the aim to contribute to peacekeeping and peacemaking. In the absence of a European army or a permanent pool of military and civilian resources for external crisis management operations, the national government institutions of EU member states are responsible for delivering the required capabilities to implement ESDP operations. This thesis asks two interrelated questions associated with this recent institutional challenge for the national administrations. Firstly, what has been the impact of ESDP on national administrative structures of EU member states, both in terms of competence allocation and coordination, and secondly, which factors account for the observed processes of (resistance to) change. The analytical framework of this thesis builds upon theoretical approaches used in the Europeanisation literature and complements the structure-based historical institutionalist argument of path dependency with an actor-based approach derived from individual learning. The thesis examines both governmental learning and political learning, and emphasises the importance of the changing preferences and beliefs of the principal political actors. Combining structure and agency, the main hypothesis offered to explain administrative change claims that individual political learning has the capacity to overcome institutional path-dependency. This analytical framework is tested in three case studies: Belgium (1999-2007), Sweden (1999-2008), and Germany (1999-2009). The thesis therefore makes both theoretical and empirical contributions to the subject of administrative adaptation to ESDP.

The construction of a multiwavelet basis in higher dimensions begins with a refinement equation of the form f(x) = ∑k∈Γc kf(Ax − k), where f : [special characters omitted], Γ is a full-rank lattice in [special characters omitted], A is a dilation matrix, and each c k is an r x r complex matrix. In one dimension, a great deal is known about how to construct wavelets with certain desirable properties, including orthogonality, symmetry, smoothness, and rapid decay. The natural setting for many applications is in higher dimensions. These higher-dimensional constructions have proved difficult because the classical one-dimensional techniques fail dramatically in these settings. This thesis focuses on the development of new time-domain based techniques not limited by the factorization requirements or other difficulties inherent in the one-dimensional approach. For many applications, it is desirable to construct a scaling function with a certain degree of smoothness. One tool for examining the smoothness of a refinable function is the joint spectral radius. It is shown that for a multivariate vector scaling function, if the joint spectral radius of a particular set of matrices is less than ρ(A)−ν , then the scaling function is ν-times continuously differentiable. In order to construct an orthonormal wavelet basis, the translates of the scaling function along Γ must form an orthonormal basis for their span. In the one-dimensional, single function setting, this can be characterized by Lawton's condition, which is stated in terms of the eigenvalues of a matrix whose entries are constructed from the refinement coefficients. Conditions analogous to Lawton's condition are developed in the multivariate setting which determine if the Γ-translates of a vector scaling function are orthonormal. Finally, the issue of constructing the wavelets from the scaling function is addressed. If the scaling function has orthonormal translates, this is equivalent to completing an orthogonal matrix formed from the refinement coefficients. In general, it is not known if it is possible to complete this matrix, or how to do so. If D is a complete set of representatives of Γ/ A(Γ), it is assumed that the nonzero refinement coefficients occur in D ∪ (D + Ai) for some nonzero i ∈ Γ. Under this assumption, a constructive algorithm is given for completing the orthogonal matrix.

\"Oh, geez, I'm sorry,\" he says. \"I gotta keep an eye on my language.\" \"Don't worry\" smiles [Vivian Parker]. \"Some porridge?\" \"At least there's only one room to clean,\" [Helen Skiba] quipped philosophically. The two couples often spend evenings together, playing cards and drinking endless cups of Helen's Swedish coffee. John and Vivian usually host as it is easier for Helen to bring the coffee than for the Parkers to bundle up the children and trudge through the snow to their place. Also, [John] and Vivian's cabin is larger and they have four chairs.