13C-metabolic flux analysis for mevalonate-producing strain of Escherichia coli.

Mevalonate (MVA) is used to produce various useful products such as drugs, cosmetics and food additives. An MVA-producing strain of Escherichia coli (engineered) was constructed by introducing mvaES genes from Enterococcus faecalis. The engineered strain produced 1.84 mmol/gDCW/h yielding 22% (C-mol/C-mol) of MVA from glucose in the aerobic exponential growth phase. The mass balance analysis revealed that the MVA yield of the engineered strain was close to the upper limit at the biomass yield. Since MVA is synthesized from acetyl-CoA using NADPH as a cofactor, the production of MVA affects central metabolism in terms of carbon utilization and NADPH requirements. The reason for this highly efficient MVA production was investigated based on 13C-metabolic flux analysis. The estimated flux distributions revealed that the fluxes of acetate formation and the TCA cycle in the engineered strain were lower than those in the control strain. Although the oxidative pentose phosphate pathway is considered as the NADPH generating pathway in E. coli, no difference of the flux was observed between the control and engineered strains. The production/consumption balance of NADPH suggested that additional requirement of NADPH for MVA synthesis was obtained from the transhydrogenase reaction in the engineered strain. Comparison between the measured flux distribution and the ideal values for MVA production proposes a strategy for further engineering to improve the MVA production in E. coli.

Estudio in vitro sobre los efectos de las estatinas en las curvas de crecimiento celular y su reversión con mevalonato / In vitro study over statins effects on cellular growth curves and its reversibility with mevalonate

Los inhibidores de la HMG-Coa-reductasa, conocidos como estatinas, son los agentes farmacológicos disponibles en el mercado que tienen un mayor efecto hipocolesterolemiante. Los ensayos clínicos y las evidencias experimentales han demostrado que las estatinas tienen un potente efecto antiaterosclerótico. Este efecto es, en parte, la consecuencia del descenso de lípidos, pero también se debe a ciertas acciones pleiotrópicas.Los llamados efectos pleiotrópicos se refieren a distintos aspectos de la función celular: inflamación, coagulación y actividad vasomotora. Estos efectos son mediados, bien indirectamente a través de la reducción del cLDL, o bien por vía directa en las funciones celulares. Aunque muchas de las acciones pleiotrópicas de las estatinas pueden ser un efecto de clase, alguna de ellas puede ser exclusiva de ciertos fármacos y mostrar diferencias en su actividad farmacológica. Así, aunque las estatinas tienen un efecto común sobre los niveles de cLDL, las diferencias en la estructura química y en el perfil farmacocinética pueden motivar variaciones en los efectos pleiotrópicos.En el presente artículo analizamos los efectos in vitro de diferentes estatinas sobre diferentes líneas celulares de células implicadas en el proceso aterogénico: células endoteliales, fibroblastos y células musculares de la pared vascular. En relación con nuestros resultados, comprobamos que los efectos de diferentes dosis de diferentes estatinas sobre las curvas de crecimiento de las diferentes líneas celulares producen diferentes efectos, con independencia de los efectos dependientes de la clase. En consecuencia, los efectos pleiotrópicos sobre el crecimiento celular y su reversibilidad con mevalonato son diferentes según la molécula y la dosis empleadas

HMG-CoA-Reductase inhibitors, also known as statins, are currently the most powerful cholesterol-lowering drugs available on the market. Clinical trials and experimental evidence suggest that statins have heavy anti-atherosclerotic effects. These are in part consequence of lipid lowering but also result from pleiotropic actions of the drugs. These so-called pleiotropic properties affect various aspects of cell function, inflammation, coagulation, and vasomotor activity. These effects are mediated either indirectly through LDL-c reduction or via a direct effect on cellular functions. Although many of the pleiotropic properties of statins may be a class effect, some may be unique to certain agents and account for differences in their pharmacological activity. So, although statins typically have similar effects on LDL-c levels, differences in chemical structure and pharmacokinetic profile can lead to variations in pleiotropic effects. In this paper we analize the in vitro effects of different statins over different cell lines from cells implicated in atherosclerotic process: endothelial cells, fibroblasts, and vascular muscular cells. In relation with our results we can proof that the effects of different dosis of different statins provides singular effects over growth curves of different cellular lines, a despite of a class-dependent effects. So, pleiotropic effects and its reversibility with mevalonate are different according with the molecule and the dosis

Distinct isoprenoid origins of cis- and trans-zeatin biosyntheses in Arabidopsis.

Plants produce the common isoprenoid precursors isopentenyl diphosphate and dimethylallyl diphosphate (DMAPP) through the methylerythritol phosphate (MEP) pathway in plastids and the mevalonate (MVA) pathway in the cytosol. To assess which pathways contribute DMAPP for cytokinin biosynthesis, metabolites from each isoprenoid pathway were selectively labeled with (13)C in Arabidopsis seedlings. Efficient (13)C labeling was achieved by blocking the endogenous pathway genetically or chemically during the feed of a (13)C labeled precursor specific to the MEP or MVA pathways. Liquid chromatography-mass spectrometry analysis demonstrated that the prenyl group of trans-zeatin (tZ) and isopentenyladenine is mainly produced through the MEP pathway. In comparison, a large fraction of the prenyl group of cis-zeatin (cZ) derivatives was provided by the MVA pathway. When expressed as fusion proteins with green fluorescent protein in Arabidopsis cells, four adenosine phosphate-isopentenyltransferases (AtIPT1, AtIPT3, AtIPT5, and AtIPT8) were found in plastids, in agreement with the idea that the MEP pathway primarily provides DMAPP to tZ and isopentenyladenine. On the other hand, AtIPT2, a tRNA isopentenyltransferase, was detected in the cytosol. Because the prenylated adenine moiety of tRNA is usually of the cZ type, the formation of cZ in Arabidopsis seedlings might involve the transfer of DMAPP from the MVA pathway to tRNA. Distinct origins of large proportions of DMAPP for tZ and cZ biosynthesis suggest that plants are able to separately modulate the level of these cytokinin species.

Urinary excretion and serum concentration of mevalonic acid during acute intake of alcohol.

The influence of 2 different alcoholic beverages containing an equal amount of alcohol (48 g), 1 with mevalonic acid (beer) and 1 without (vodka), on the urinary excretion and serum concentration of mevalonic acid was investigated in 7 healthy subjects. Drinking 1 L of beer at night containing 608 microg/L mevalonic acid more than doubled the urinary excretion of mevalonic acid the following 12 hours, on average from 103 +/- 15 microg/12 h to 211 +/- 17 microg/12 h (P < .001; 18% of the administered dose). Drinking the same amount of alcohol as vodka had no effect, but urinary mevalonic acid output increased slightly the following day (7 AM to 7 PM) after ingestion of both alcoholic beverages. Serum concentrations of mevalonic acid were significantly increased the following morning after ingestion of beer (from 3.22 +/- 0.20 ng/mL to 6.79 +/- 0.58 ng/mL) or vodka (from 3.23 +/- 0.37 ng/mL to 5.36 +/- 0.55 ng/mL, P < .002 for both). An increase in the ratio of lathosterol to cholesterol in serum, another indicator of 3beta-hydroxy-3beta-methylglutaryl coenzyme A reductase activity in the liver, was also observed (+18% and +25%, respectively). After oral administration of [13C2] mevalonic acid at night, 20% +/- 0.7% of the dose was excreted in urine the following 12 hours, and only trace amounts thereafter. No [13C2] mevalonic acid could be detected in serum the following morning. We conclude that the absorption of dietary mevalonic acid and alcohol-induced mevalonic acid synthesis affects the urinary excretion and serum concentration of this cholesterol precursor. Therefore, studies using mevalonic acid as a marker of cholesterol synthesis must be carefully monitored regarding dietary mevalonic acid intake and alcohol consumption.

Chloroplastic prenylated proteins.

By in vivo [3H]mevalonate labelling of spinach combined with biochemical analysis, evidence is provided for the existence of protein prenylation in chloroplasts. Approximately 20 prenylated polypeptides were resolved by SDS-PAGE followed by autoradiography. Thermolysin treatment of intact chloroplasts revealed that about 40% of the prenylated polypeptides were associated with the cytoplasmic surface of the outer envelope membrane. The remaining portion was present in thylakoids and/or the inner envelope membrane. The majority of the prenylated polypeptides were associated with larger membrane protein complexes. A farnesyl protein transferase activity was found to be associated with the thylakoid membrane.

Intestinal brush-border membrane transport of monocarboxylic acids mediated by proton-coupled transport and anion antiport mechanisms.

Intestinal brush-border membrane transport of monocarboxylic acids was investigated by using rabbit intestinal brush-border membrane vesicles (BBMVs) and isolated intestinal tissues mounted on Ussing-type chambers. [3H]Mevalonic acid uptake by BBMVs showed an overshoot phenomenon in the presence of an inwardly directed proton gradient, but not in the presence of an inwardly directed sodium gradient or an outwardly directed HCO3- or chloride gradient. Initial uptake of mevalonic acid was saturable in the presence of a proton gradient. Uptake of [3H]mevalonic acid was inhibited by various monocarboxylic acids, including acetic acid, benzoic acid, lactic acid, nicotinic acid, pravastatin, salicylic acid and valproic acid, but not by dicarboxylic acid or amino acids. Acetic acid, which is transported by both anion antiport and proton-coupled transport systems, induced serosal bicarbonate-dependent alkalinization in the mucosal-side bathing solution of rabbit jejunal tissues, when examined in Ussing-type chambers. Pravastatin, which is a structural analogue of mevalonic acid and is absorbed via proton-coupled transport like mevalonic acid, did not. The result demonstrates that acetic acid is transported by the bicarbonate-dependent anion antiport system, whereas pravastatin is not. So, it is suggested that monocarboxylic acids are transported by at least two independent transporters, namely, a proton-coupled transporter for most monocarboxylic acids, including mevalonic acid, pravastatin and acetic acid, and an anion antiporter for acetic acid, but not for mevalonic acid or pravastatin. Activation of anion antiporter can induce HCO3- secretion in intact intestine.

In vitro and in vivo synthesis of dolichol and other main mevalonate products in various organs of the rat.

The relative rate of biosynthesis of dolichol from [3H]mevalonate in nine rat organs was studied in slices and in the whole animal. This biosynthesis was also compared to that of cholesterol and ubiquinone. All tissues examined are able to synthesize dolichol, as well as ubiquinone and cholesterol. Comparison of the data from slices in vitro with the in vivo studies demonstrated relatively good agreement for dolichol and ubiquinone synthesis. Although dolichol of high specific radioactivity was recovered in the blood, redistribution between organs, such as occurs with cholesterol, appears to be insignificant. The highest rates of dolichol biosynthesis were found in kidney, spleen and liver. On the other hand, muscle makes the largest contribution to total body dolichol synthesis. Newly synthesized dolichol also appears in the bile, but excretion by this route is far from sufficient to account for dolichol turnover. Incorporation of mevalonate into the final products is mainly dependent on biosynthetic activity. For comparison of the biosynthetic rates in different organs, possible sources of errors (such as variations in the size of the precursor pool, limitation by the rate of precursor uptake or non-linear incorporation) were investigated the size of the mevalonate pool in various organs. Equilibration of this pool with exogenous mevalonate is a rapid and passive process. The size of the mevalonate pool does not determine the rates of cholesterol and dolichol biosynthesis, indicating the presence of regulatory steps in the terminal portion of these biosynthetic pathways.