Biomedical subjects

Fernando Alvarez-Vasquez

Publications and source records attributed to Fernando Alvarez-Vasquez.

5 recordsLinked to original sources

PubMed2006

Necessary role for the Lag1p motif in (dihydro)ceramide synthase activity.

Lag1 (longevity assurance gene 1) homologues, a family of transmembrane proteins found in all eukaryotes, have been shown to be necessary for (dihydro)ceramide synthesis. All Lag1 homologues contain a highly conserved stretch of 52 amino acids known as the Lag1p motif. However, the functional significance of the conserved Lag1p motif for (dihydro)ceramide synthesis is currently unknown. In this work, we have investigated the function of the motif by introducing eight point mutations in the Lag1p motif of the mouse LASS1 (longevity assurance homologue 1 of yeast Lag1). The (dihydro)ceramide synthase activity of the mutants was tested using microsomes in HeLa cells and in vitro. Six of the mutations resulted in loss of activity in cells and in vitro. In addition, our results showed that C18:0 fatty acid CoA (but not cis-C18:1 fatty acid CoAs) are substrates for LASS1 and that LASS1 in HeLa cells is sensitive to fumonisin B1, an in vitro inhibitor of (dihydro)ceramide synthase. Moreover, we mutated the Lag1p motif of another Lag homologue, human LASS5. The amino acid substitutions in the human LASS5 were the same as in mouse LASS1, and had the same effect on the in vitro activity of LASS5, suggesting the Lag1p motif appears to be essential for the enzyme activity of all Lag1 homologues.

Amino Acid Sequence↗

PubMed2005

Simulation and validation of modelled sphingolipid metabolism in Saccharomyces cerevisiae.

Mathematical models have become a necessary tool for organizing the rapidly increasing amounts of large-scale data on biochemical pathways and for advanced evaluation of their structure and regulation. Most of these models have addressed specific pathways using either stoichiometric or flux-balance analysis, or fully kinetic Michaelis-Menten representations, metabolic control analysis, or biochemical systems theory. So far, the predictions of kinetic models have rarely been tested using direct experimentation. Here, we validate experimentally a biochemical systems theoretical model of sphingolipid metabolism in yeast. Simulations of metabolic fluxes, enzyme deletion and the effects of inositol (a key regulator of phospholipid metabolism) led to predictions that show significant concordance with experimental results generated post hoc. The model also allowed the simulation of the effects of acute perturbations in fatty-acid precursors of sphingolipids, a situation that is not amenable to direct experimentation. The results demonstrate that modelling now allows testable predictions as well as the design and evaluation of hypothetical 'thought experiments' that may generate new metabolomic approaches.

Computer Simulation↗

PubMed2004

Integration of kinetic information on yeast sphingolipid metabolism in dynamical pathway models.

For the first time, kinetic information from the literature was collected and used to construct integrative dynamical mathematical models of sphingolipid metabolism. One model was designed primarily with kinetic equations in the tradition of Michaelis and Menten whereas the other two models were designed as alternative power-law models within the framework of Biochemical Systems Theory. Each model contains about 50 variables, about a quarter of which are dependent (state) variables, while the others are independent inputs and enzyme activities that are considered constant. The models account for known regulatory signals that exert control over the pathway. Standard mathematical testing, repeated revisiting of the literature, and numerous rounds of amendments and refinements resulted in models that are stable and rather insensitive to perturbations in inputs or parameter values. The models also appear to be compatible with the modest amount of experimental experience that lends itself to direct comparisons. Even though the three models are based on different mathematical representations, they show dynamic responses to a variety of perturbations and changes in conditions that are essentially equivalent for small perturbations and similar for large perturbations. The kinetic information used for model construction and the models themselves can serve as a starting point for future analyses and refinements.

Models, Biological↗

PubMed2004

Analysis of dynamic labeling data.

Comprehensive assessments of the organization and regulation of metabolic pathways cannot be limited to steady-state measurements alone but require dynamic time series data. One experimental means of generating such data consists of radioactively labeling precursors and measuring their fate over time. While labeling experiments belong to the standard repertoire of biological laboratory techniques, corresponding mathematical tools for analyzing the non-linear dynamics of tracers are scarce. The article addresses this issue, using Biochemical Systems Theory as the modeling framework. The description of the dynamics of labeled metabolites alone is difficult, but it is demonstrated that these difficulties are easily overcome by setting up dynamic models in two or three blocks, one for the kinetics of the total pools, the second just for the labeled portions, and the third, optional, block for the remaining unlabeled components. Since the dynamic model is not limited in complexity and can account for linear pathways, converging and diverging branches, cycles, and the various observed modes of regulation, the proposed method of non-linear tracer analysis is rather general and permits simulations of most standard labeling experiments, both at steady state and during transients.

Computer Simulation↗

PubMed2002

Modeling, optimization and experimental assessment of continuous L-(-)-carnitine production by Escherichia coli cultures.

In a previous paper Cánovas et al. (Biotechnol Bioeng 2002;77:764-775) presented a model for L-(-)-carnitine production using Escherichia coli O44 K74, in a cell-recycle bioreactor for the biotransformation of crotonobetaine into L-carnitine. In this work we optimize this biotechnological setup and experimentally verify the predicted optimal parameter profiles. Provided with a reliable and robust S-system description of the cell-bioreactor combined system, we applied the Indirect Optimization Method described by Torres et al. (Biotechnol Bioeng 1997;55(5):758-772; Food Technol Biotechnol 1998;36(3):177-184). This optimization approach provides different parameter value profiles, all of which are compatible with the cell physiology and the bioreactor operating conditions, that yield increased rates of L-(-)-carnitine production. Three parameters were seen to be of critical importance for maximizing L-(-)-carnitine production: the dilution rate, the initial crotonobetaine concentration, and the carnitine dehydratase activity. When the first two were changed in the experimental setup, there was a 74% increase in the L-(-)-carnitine production rate, performance that was in close agreement with the predictions of the model. In accordance with the optimized solution, a further improvement (90% increase in the L-(-)-carnitine production rate) could be attained by over-expressing up to 5 times the carnitine dehydratase basal activity. Thus the optimization approach shown herein provides experimental evidence of a new strategy which demonstrates the possible variables that can be subjected to modifications compatible with the cell physiology and bioreactor operating conditions, and which are able to yield increased rates of L-(-)-carnitine production.

Alcohol Oxidoreductases↗