Metabolic vectors

Modified mammalian cell systems for the study of the role of transporters and/or metabolism in oral absorption consists of two main components the cell line and the vector bearing a cDNA encoding the protein of interest. The cell line serves two roles first, to support adequate expression of the cDNA and second, to provide a barrier function which is generally critical in assays for transporter function. 

Further work at EniTecnologies was conducted with Rhodococcus strains. Rhodococ-cus was selected for its metabolical versatility, easy availability in soils and water, and remarkable solvent tolerance. Its capabilities for catalyzing diverse transformation reactions of crude oils, such as sulfur removal, alkanes and aromatics oxidation and catabolism caught their attention. Hence, genetic tools for the engineering of Rhodococcus strains have been applied to improve its biotransformation performance and its tolerance to certain common contaminants of the crude oil, such as cadmium. The development of active biomolecules led to the isolation and characterization of plasmid vectors and promoters. Strains have been constructed in which the careful over-expression of selected components of the desulfurization pathway leads to the enhancement of the sulfur removal activity in model systems. Rhodococcus, Gordona, and Nocardia were transformed in this way trying to improve their catalytic performance in BDS. In a... 

The genes corresponding to the enzymes involved in conversion of benzothiophene from the claimed organisms as well as the vectors carrying the genes were protected in the patent. The details of the metabolic pathway were given in Section 2.2.3, the proposed metabolic pathway is shown here for completion purposes (Fig. 2). 

The majority of viruses that infect plants have single-stranded, positive-sense RNA genomes. It has therefore been necessary to use infectious cDNA clones for the in vitro manipulation of RNA viruses, allowing them to be developed as effective tools for the commercial production of target proteins in plants. This approach has also been used to study the genetic and metabolic profiles of both viruses and their host plants. Siegel [14] conceptualized the potential use of RNA viruses as expression vectors. Brome mosaic virus (BMV) and Tobacco mosaic vims (TMV) were the first two RNA viruses to be converted into expression vectors. These vectors have since been pro-... 

Ye, X., Robinson, M. B., Batshaw, M. L., Furth, E. E., Smith, I. and Wilson, J. M. Prolonged metabolic correction in adult ornithine transcarbamylase-deficient mice with adenoviral vectors. / Biol. Chem. 271 3639-3646,1996. 

We seek to describe the time-dependent behavior of a metabolic network that consists of m metabolic reactants (metabolites) interacting via a set of r biochemical reactions or interconversions. Each metabolite S, is characterized by its concentration 5,(f) > 0, usually measured in moles/volume. We distinguish between internal metabolites, whose concentrations are affected by interconversions and may change as a function of time, and external metabolites, whose concentrations are assumed to be constant. The latter are usually omitted from the m-dimensional time-dependent vector of concentrations S(t) and are treated as additional parameters. If multiple compartments are considered, metabolites that occur in more than one compartments are assigned to different subscripts within each compartment. 

An analysis of the right nullspace K provides the conceptual basis of flux balance analysis and has led to a plethora of highly successful applications in metabolic network analysis. In particular, all steady-state flux vectors v° = v(S°,p) can be written as a linear combination of columns Jfcx- of K, such that... 

Aiming at a network-based pathway analysis, the elementary flux modes provide a handle on the set of possible pathways through a metabolic network. In particular, each feasible steady-state flux distribution can be represented by a nonnegative combination of generating vectors that span the flux cone defined... 

Figure 26. The proposed workflow of structural kinetic modeling Rather than constructing a single kinetic model, an ensemble of possible models is evaluated, such that the ensemble is consistent with available biological information and additional constraints of interest. The analysis is based upon a (thermodynamically consistent) metabolic state, characterized by a vector S° and the associated flux v° v(S°). Since based only on the an evaluation of the eigenvalues of the Jacobian matrix are evaluated, the approach is (computationally) applicable to large scale system. Redrawn and adapted from Ref. 296.

In contrast, SKM does not assume knowledge of thespecific functional form of the rate equations. Rather, the system is evaluated in terms of generalized parameters, specified by the elements of the matrices A and 0X. In this sense, the matrices A and 0 x are bona fide parameters of the system The pathway is described in terms ofan average metabolite concentration S°, and a steady-state flux vector v°, together defining the metabolic state of the pathway. Additionally, we assume that the substrate only affects reaction v2, the saturation matrix is thus fully specified by a single parameter Of 6 [0,1], Note that the number of parameters is identical to the number used within the explicit equation. The structure of the parameter matrices is... 

Transfection ofmouse SlPphosphatase into HEK 293 cells, resulting in a 3-fold increase in membrane SIP phosphatase activity, caused a 50% deaease in SIP levels (reduced by 0.6 pmol/nmol phospholipid) and a 2 fold inaease in ceramide (inaeased by 23 pmol/nmol phospholipid) whereas sphingosine levels were similar to vector controls (0.8 pnnol/nmol phosphohpid). SIP phosphatase transfected cells underwent apoptosis in response to serum withdrawal, C2-ceramide, peroxide or doxorubicin with 2-3 fold higher frequency compared to vector-transfected control cells. Surprisingly, exogenously added SIP, which normally confers protechon, inaeased apoptosis. This may be due to its metabolism to ceramide (Mandala et al, 2000) although other factors may also be involved. 

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