Zeaxanthin and menaquinone-7 biosynthesis in Sphingobacterium multivorum via the methylerythritol phosphate pathway.

Feeding of [1-(13)C]glucose, [U-(13)C(6)]glucose, [3-(13)C]alanine and [1-(13)C]acetate to Sphingobacterium multivorum showed that this bacterium utilizes the methylerythritol phosphate pathway for the biosynthesis of menaquinone-7 and zeaxanthin, a carotenoid of industrial importance. Differential incorporation of the labeled precursors gave some insight into the preferred carbon sources involved in isoprenoid biosynthesis.

Beta(3)-adrenoceptor agonist-induced increases in lipolysis, metabolic rate, facial flushing, and reflex tachycardia in anesthetized rhesus monkeys.

The effects of two beta(3)-adrenergic receptor agonists, (R)-4-[4-(3-cyclopentylpropyl)-4,5-dihydro-5-oxo-1H-tetrazol-1-yl]-N-[4-[2-[[2-hydroxy-2-(3-pyridinyl)ethyl]amino]ethyl]phenyl]benzenesulfonamide and (R)-N-[4-[2-[[2-hydroxy-2-(3-pyridinyl)- ethyl]amino]ethyl]phenyl]-1-(4-octylthiazol-2-yl)-5-indolinesulfonamide, on indices of metabolic and cardiovascular function were studied in anesthetized rhesus monkeys. Both compounds are potent and specific agonists at human and rhesus beta(3)-adrenergic receptors. Intravenous administration of either compound produced dose-dependent lipolysis, increase in metabolic rate, peripheral vasodilatation, and tachycardia with no effects on mean arterial pressure. The increase in heart rate in response to either compound was biphasic with an initial rapid component coincident with the evoked peripheral vasodilatation and a second more slowly developing phase contemporaneous with the evoked increase in metabolic rate. Because both compounds exhibited weak binding to and activation of rhesus beta(1)-adrenergic receptors in vitro, it was hypothesized that the increase in heart rate may be reflexogenic in origin and proximally mediated via release of endogenous norepinephrine acting at cardiac beta(1)-adrenergic receptors. This hypothesis was confirmed by determining that beta(3)-adrenergic receptor agonist-evoked tachycardia was attenuated in the presence of propranolol and in ganglion-blocked animals, under which conditions there was no reduction in the evoked vasodilatation, lipolysis, or increase in metabolic rate. It is not certain whether the beta(3)-adrenergic receptor-evoked vasodilatation is a direct effect of compounds at beta(3)-adrenergic receptors in the peripheral vasculature or is secondary to the release or generation of an endogenous vasodilator. Peripheral vasodilatation in response to beta(3)-adrenergic receptor agonist administration was not attenuated in animals administered mepyramine, indomethacin, or calcitonin gene-related peptide(8-37). These findings are consistent with a direct vasodilator effect of beta(3)-adrenergic receptor agonists.

Farnesol-induced cell death and stimulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in tobacco cv bright yellow-2 cells.

Growth inhibition of tobacco (Nicotiana tabacum L. cv Bright Yellow-2) cells by mevinolin, a specific inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) could be partially overcome by the addition of farnesol. However, farnesol alone inhibited cell division and growth as measured by determination of fresh weight increase. When 7-d-old tobacco cv Bright Yellow-2 cells were diluted 40-fold into fresh culture, the cells exhibited a dose-dependent sensitivity to farnesol, with 25 microM sufficient to cause 100% cell death, as measured by different staining techniques, cytometry, and monitoring of fragmentation of genomic DNA. Cells were less sensitive to the effects of farnesol when diluted only 4-fold. Farnesol was absorbed by the cells, as examined by [1-(3)H]farnesol uptake, with a greater relative enrichment by the more diluted cells. Both mevinolin and farnesol treatments stimulated apparent HMGR activity. The stimulation by farnesol was also reflected in corresponding changes in the steady-state levels of HMGR mRNA and enzyme protein with respect to HMGR gene expression and enzyme protein accumulation.

Expression of Brassica juncea 3-hydroxy-3-methylglutaryl CoA synthase is developmentally regulated and stress-responsive.

3-Hydroxy-3-methylglutaryl-coenzyme A synthase (HMGS) is an enzyme in mevalonate biosynthesis. In plants, investigations have focused on HMG CoA reductase (HMGR) and less is known of the preceding enzyme, HMGS. To understand the regulation of HMGS, we have isolated a Brassica juncea cDNA encoding HMGS, BjHMGS1, for use as a hybridization probe in Northern blot analyses. BjHMGS is expressed in all plant organs and shows developmental regulation in flower, seed and seedling, with highest expression in early development. In seedlings, expression is highest in young hypocotyls and is induced during the greening of etiolated cotyledons. BjHMGS is down-regulated by abscisic acid, osmotic stress and dehydration, the effects of which arrested seedling growth. Thus BjHMGS expression shows correlation with rapid cell division and growth, like HMGR. This is not unexpected, as mevalonate is the precursor to many essential isoprenoid compounds, including sterols for membrane biogenesis. Wounding, methyl jasmonate or salicylic acid induce BjHMGS expression, suggesting that, like HMGR, HMGS is involved in defence. As in animals, coordinated regulation of HMGS with HMGR occurred in B. juncea upon germination and in response to salicylic acid. HMGS assays confirmed that Escherichia coli-expressed recombinant BjHMGS1 shows HMGS activity that is inhibited by F244, a specific inhibitor of HMGS. Southern blot analysis revealed gene families encoding HMGS in Brassica species and a summation of homologous genes in the fusion amphidiploid genome of B. juncea, a bi-parental species derived from diploids B. nigra and B. campestris.

Metabolism of farnesyl diphosphate in tobacco BY-2 cells treated with squalestatin.

Plant isoprenoids represent a large group of compounds with a wide range of physiological functions. In the cytosol, isoprenoids are synthesized via the classical acetate/mevalonate pathway. In this pathway, farnesyl diphosphate (FPP) occupies a central position, from which isoprene units are dispatched to the different classes of isoprenoids, with sterols as the major end products. The present work deals with effects of squalestatin (SQ) on the metabolism of FPP in proliferating and synchronized cultured tobacco cv. Bright Yellow-2 cells. SQ is a potent inhibitor of squalene synthase (SQS), the first committed enzyme in the sterol pathway. At nanomolar concentrations, SQ severely impaired cell growth and sterol biosynthesis, as attested by the rapid decrease in SQS activity. At the same time, it triggered a several-fold increase in both the enzymic activity and mRNA levels of 3-hydroxy-3-methylglutaryl CoA reductase. When SQ was added to cells synchronized by aphidicolin treatment, it was found to block the cell cycle at the end of G(1) phase, but no cell death was induced. Tobacco cells were also fed exogenous tritiated trans-trans farnesol, the allylic alcohol derived from FPP, in the presence and absence of SQ. Evidence is presented that this compound was incorporated into sterols and ubiquinone Q(10). In the presence of SQ, the sterol pathway was inhibited, but no increase in the radioactivity of ubiquinone was observed, suggesting that this metabolic channel was already saturated under normal conditions.

A selective human beta3 adrenergic receptor agonist increases metabolic rate in rhesus monkeys.

Activation of beta3 adrenergic receptors on the surface of adipocytes leads to increases in intracellular cAMP and stimulation of lipolysis. In brown adipose tissue, this serves to up-regulate and activate the mitochondrial uncoupling protein 1, which mediates a proton conductance pathway that uncouples oxidative phosphorylation, leading to a net increase in energy expenditure. While chronic treatment with beta3 agonists in nonprimate species leads to uncoupling protein 1 up-regulation and weight loss, the relevance of this mechanism to energy metabolism in primates, which have much lower levels of brown adipose tissue, has been questioned. With the discovery of L-755,507, a potent and selective partial agonist for both human and rhesus beta3 receptors, we now demonstrate that acute exposure of rhesus monkeys to a beta3 agonist elicits lipolysis and metabolic rate elevation, and that chronic exposure increases uncoupling protein 1 expression in rhesus brown adipose tissue. These data suggest a role for beta3 agonists in the treatment of human obesity.

Mevalonate-derived isopentenyl diphosphate is the biosynthetic precursor of ubiquinone prenyl side chain in tobacco BY-2 cells.

Study of the incorporation of 13C-labelled glucose or pyruvate into the isoprenoids of tobacco BY-2 cells allowed the biosynthetic origin of isopentenyl diphosphate to be determined. Sterols synthesized in the cytoplasm and the prenyl chain of ubiquinone Q10 located in mitochondria were derived from the same isopentenyl diphosphate pool, synthesized from acetyl-CoA through mevalonate, whereas the prenyl chain of plastoquinone was obtained from the mevalonate-independent glyceraldehyde 3-phosphate/pyruvate route, like all chloroplast isoprenoids from higher plants. These results are in accord with the compartmentation and complete enzymic independence of the biosynthesis of long-chain all-trans polyprenols in mitochondria and chloroplasts.

Effects of mevinolin on cell cycle progression and viability of tobacco BY-2 cells.

Mevinolin, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, was used to study the importance of mevalonic acid (MVA) for cell cycle progression of tobacco (Nicotiana tabacum L.) BY-2 cells. After treatment with 5 microM mevinolin, the cell cycle progression was completely blocked and two cell populations accumulated (80% in phase G0/G1 and 20% in G2/M). The arrest could be released by subsequent addition of MVA. Effects were compared to those caused by aphidicolin, an inhibitor of alpha-like DNA polymerases that blocks cell cycle at the entry of the S phase. The 80% proportion of mevinolin-treated TBY-2 cells was clearly arrested before the aphidicolin-inducible block. By the aid of a double-blocking technique, it was shown that the mevinolin-induced cell arrest of highly synchronized cells was due to interaction with a control point located at the mitotic telophase/entry G1 phase. Depending on the developmental stage, mevinolin induced rapid cell death in a considerable percentage of cells. Mevinolin treatment led to a partial synchronization, as shown by the increase in mitotic index. The following decrease was correlated with the above-mentioned induction of cell death.

Cloning of a cDNA encoding cytosolic acetoacetyl-coenzyme A thiolase from radish by functional expression in Saccharomyces cerevisiae.

A cDNA coding for radish (Raphanus sativus L.) acetoacetyl-coenzyme A thiolase (AACT) was cloned by complementation of the erg10 mutation affecting AACT in yeast (Saccharomyces cerevisiae). The longest reading frame encodes a protein of 406 amino acids with a predicted relative molecular weight of 42,032, with significant similarities to eukaryotic and prokaryotic thiolases. There is no evidence for the presence of a leader peptide characteristic, e.g. of glyoxysomal thiolase. Yeast transformants expressing the radish AACT gene placed under the control of the GAL1 promoter exhibited a 10-fold higher enzyme activity than a wild-type yeast strain after induction by galactose. This enzyme activity is exclusively localized in the soluble fraction but not in membranes. These data indicate that we have cloned a gene encoding cytoplasmic (biosynthetic) AACT. Genomic DNA gel blot analysis suggests the presence of a single AACT gene, which is expressed in all parts of the seedling. Expression in cotyledons appears to be light-stimulated. We present preliminary evidence that a smaller transcript represents an antisense species being read from the same gene.

Cloning of Arabidopsis thaliana glutathione synthetase (GSH2) by functional complementation of a yeast gsh2 mutant.

Glutathione (L-gamma-glutamyl-L-cysteinylglycine, GSH) plays an important role in the protection of plants against various types of stress caused by reactive oxygen species, gazeous pollutants, heavy metals and xenobiotics. A cDNA fragment containing the entire coding unit for glutathione synthetase (GSH2) of Arabidopsis thaliana was cloned by complementation of the methylglyoxal sensitivity of a gsh2 mutant of the yeast Saccharomyces cerevisiae. The cDNA encodes a protein of 478 amino acids (deduced Mr: 53783), bearing clear sequence similarities to GSH2 products from frog embryos (Xenopus laevis), rat kidney (Rattus norvegicus) and from the fission yeast (Schizosaccharomyces pombe). A highly conserved glycine-rich domain close to the carboxy-terminus was found in the GSH2 product and appears to be typical for eukaryotic glutathione synthetases. The Mr is similar to those of soluble animal enzymes, suggesting that the Arabidopsis gene also codes for a cytosolic protein. Genomic DNA-blot analysis indicates the presence of a single GSH2 gene. The yeast gsh2 mutant becomes resistant to methylglyoxal and cadmium after transformation with the plasmid bearing the Arabidopsis GSH2 cDNA. Moreover, this increased resistance is correlated to the restoration of GSH content from below detectability in mutants to about 50% of the wild-type levels in transformed cells.

Some new aspects of isoprenoid biosynthesis in plants--a review.

Plants are capable of synthesizing a myriad of isoprenoids and prenyl lipids. Much attention has been focused on 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the enzyme that synthesizes mevalonate and is generally considered responsible for the regulation of substrate flux to isoprenoids. In contrast to vertebrates, where there seems to exist only one HMGR gene, in plants a small family of isogenes appears differentially expressed in regard to location and time. Much less is known in plants about the preceding steps, viz. the conversion of acetyl-CoA to HMG-CoA. An enzyme system has been isolated from radish that can catalyze this transformation, and which shows some unusual properties in vitro. The intracellular localization of the early steps of isoprenoid biosynthesis in plant cells is still a matter of debate. The various observations and hypotheses derived from incorporation and inhibition studies are somewhat contradictory, and an attempt is being made to rationalize various findings that do not at first seem compatible. There are good arguments in favor of an exclusively cytoplasmic formation of isopentenyl pyrophosphate (IPP) via mevalonic acid, but other studies and observations suggest an independent formation in plastids. Other possibilities are being considered, such as the existence of independent (compartmentalized) biosynthetic pathways of IPP formation via the so-called Rohmer pathway. Substrate channeling through the formation of end product-specific multienzyme complexes (metabolons) with no release of substrate intermediates will also be discussed.

Lipopolysaccharide-induced hypotension and vascular hyporeactivity in the rat: tissue analysis of nitric oxide synthase mRNA and protein expression in the presence and absence of dexamethasone, NG-monomethyl-L-arginine or indomethacin.

The role of inducible nitric oxide synthase (iNOS) was examined in the hypotension and vascular hyporesponsiveness to norepinephrine (NE) invoked by lipopolysaccharide (LPS) in pentobarbital-anesthetized rats. Saline, dexamethasone (DEX), NG-monomethyl-L-arginine (LNMMA) or indomethacin (IND) were administered either pre-LPS (0.5 hr) or post-LPS (4.5 hr) treatment. Rats were then challenged with NE 10 min before LPS injection and 1, 4, and 5 hr after LPS. Administration of LPS produced a biphasic hypotension: an immediate hypotension, which partially recovered within 15 min and was unaffected by any of the pretreatments; and a secondary, more prolonged hypotension which was attenuated by DEX, LNMMA and IND. The NE-induced pressor effects were significantly attenuated 1, 4 and 5 hr post LPS. Pretreatment with LNMMA or DEX significantly attenuated the LPS-induced NE hyporesponsiveness 4 and 5 hr post LPS. LNMMA was the only post-LPS treatment able to reverse the NE hyporesponsiveness. The LPS-induced iNOS mRNA and protein expression was demonstrated in the liver, lung, spleen, heart, kidney and brain by Northern hybridization and Western blot analyses. Low levels of neuronal constitutive NOS mRNA and endothelial cell constitutive NOS mRNA were only detected in brain or myocardial tissue, respectively. Significant induction of iNOS mRNA and protein expression was also observed in the liver, lung and spleen of rats pretreated with DEX, LNMMA or IND. The continued expression of iNOS in the presence of a pharmacologically relevant dose of DEX suggests that DEX may not be an optimal pharmacological agent for defining the in vivo roles of iNOS.(ABSTRACT TRUNCATED AT 250 WORDS)

Conversion of acetyl-coenzyme A into 3-hydroxy-3-methylglutaryl-coenzyme A in radish seedlings. Evidence of a single monomeric protein catalyzing a FeII/quinone-stimulated double condensation reaction.

We solubilized from radish membranes and purified to apparent homogeneity a monomeric protein (55.5 kDa) capable of catalyzing the two-step conversion of acetyl-CoA into 3-hydroxy-3-methylglutaryl(HMG)-CoA. Unlike the situation described for other eukaryotes (yeast, animals), both enzyme activities needed for HMG-CoA synthesis (acetoacetyl-CoA thiolase, AACT and HMG-CoA synthase, HMGS) appear to be localized on a single polypeptide. Thus, the enzyme system is further referred to as AACT/HMGS. The reaction as catalyzed by purified AACT/HMGS is strongly stimulated in vitro in presence of FeII-chelates (namely EDTA) and of quinone cofactors with pyrroloquinoline quinone (PQQ) being by far the most effective one studied so far. Whereas the FeII stimulation is apparently due to a Vmax effect, PQQ increases the affinity of the enzyme system towards acetyl-CoA (1.9 microM vs. 5.9 microM, at 50 microM FeII, 100 microM EDTA, 20 microM PQQ). Stimulation by naphthoquinone (NQ) can be overcome in the presence of halogenated NQ-derivatives, while activation by PQQ remains unaffected, possibly indicating a much more specific-binding of the latter cofactor. Gel filtration experiments of enzyme after preincubation in presence of PQQ indicate that there is no covalent-binding of the quinone cofactor to the enzyme. As is also shown with partially purified enzyme from maize membranes, phenylhydrazine, known to react with PQQ as the prosthetic group of quinoproteins (see van der Meer et al. (1987) FEBS Lett. 221, 299-304), efficiently inhibits the reaction. The data lead us to suggest a reaction mechanism that involves radical formation by the redox couple FeII/PQQ, thereby possibly facilitating the energetically unfavorable Claisen condensation as catalyzed during the first partial (AACT) reaction.

MK 287: a potent, specific, and orally active receptor antagonist of platelet-activating factor.

MK 287 (L-680,573), a tetrahydrofuran analog, potently inhibited [3H]C18-PAF binding to human platelet, polymorphonuclear leukocyte (PMN) and lung membranes with K1 values of 6.1 +/- 1.5, 3.2 +/- 0.7, and 5.49 +/- 2.3 nM, respectively. The inhibitory effects are stereospecific and competitive. The racemate, L-668,750 is less potent and the enantiomer, L-680,574 is 20-fold less potent than MK 287. Inhibition of the binding of [3H]C18-PAF to human PMN membranes by MK 287 was associated with the reduction of the affinity of the radioligand but not the number of the receptor sites. Binding of other radioligands (e.g., LTB4, LTC4, C5a, FMLP) to their specific receptors was unaltered at 1-10 microM MK 287. [3H]MK 287 bound to membranes from human platelets and PMNs: KD = 2.1 +/- 0.6 and 2.9 +/- 1.2 nM, respectively. When examined on isolated human cells, MK 287 potently and selectively inhibited PAF-induced aggregation of platelets in plasma (ED50 = 56 +/- 38 nM) or gel-filtered platelets (ED50 = 1.5 +/- 0.5 nM) and elastase release from PMNs (ED50 = 4.4 +/- 2.6 nM). In studies in vivo, MK 287 inhibited PAF-induced lethality in mice (ED50 = 0.8 mg/kg orally) and PAF-induced bronchoconstriction in guinea pigs (ED50 = 0.18 mg/kg intraduodenally and 0.19 mg/kg intravenously). Inhibition of PAF-induced bronchoconstriction was accompanied by parallel rightward shifts in concentration-response curves for PAF-induced platelet aggregation measured ex vivo.

Regulatory role of microsomal 3-hydroxy-3-methylglutaryl-coenzyme A reductase in a tobacco mutant that overproduces sterols.

In a tobacco mutant callus, containing up to tenfold more sterols than the wild-type genotype, HMG-CoA reductase activity is increased by a factor of approximately three, as is the case in mutant seedlings and plants. The rate of HMG-CoA synthesis from acetyl-CoA by the coupled enzyme system acetoacetyl-CoA thiolase/HMG-CoA synthase, as well as its conversion to acetyl-CoA plus acetoacetate by action of HMG-CoA lyase are not affected. These results confirm the key-regulating role of HMG-CoA reductase in sterol biosynthesis, which seems not to be confined only to the animal kingdom, but can also be extended to plants.

Aspects related to mevalonate biosynthesis in plants.

We purified and characterized a membrane-associated enzyme system from radish (Raphanus sativus L.) that is capable of converting acetyl-CoA into 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA). The enzyme system apparently comprises acetoacetyl-CoA thiolase (EC 2.3.1.9) and HMG-CoA synthase (EC 4.1.3.5). Its activity in vitro can be strongly stimulated by FeII. When ferrous ions are applied chelated with ethylenediaminetetraacetate, citrate or adenosine 5-triphosphate (ATP), the stimulation is further increased. Stimulation is due to a higher catalytic efficiency as indicated by an increase in Vmax, whereas the affinity of the enzyme towards acetyl-CoA remains constant (Km = 6 micro M). A considerable portion of HMG-CoA lyase activity is associated with the same membranes. HMG-CoA lyase (EC 4.1.3.4) is also solubilized and partially co-purified. Its activity requires comparatively high concentrations of Mg2+. The conversion of HMG-CoA to mevalonic acid is catalyzed by HMG-CoA reductase (EC 1.1.1.34) that is associated with the same membranes. By cDNA encoding the Arabidopsis HMG-CoA reductase, we isolated a corresponding gene from a cDNA library newly established from etiolated radish seedlings. This full-length cDNA, referred to as lambda cRS3, encodes a polypeptide 583 amino acids with a molecular mass of about 63 kDa. The hydropathy profile suggests the presence of two hydrophobic membrane-spanning domains within the N-terminal 165 amino acids. The carboxy-terminal part, where the catalytic site resides, is highly conserved in all eukaryotic HMG-CoA reductase genes sequenced so far.

Expression of catalytically active radish 3-hydroxy-3-methylglutaryl coenzyme A reductase in Escherichia coli.

Two fragments of a cDNA encoding radish 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) were cloned into the vector pET-8c and expressed in Escherichia coli. The large fragment, encoding both the membrane and the cytosolic domains, was expressed at low level, essentially as an insoluble protein without enzymatic activity. In contrast, the fragment encoding only the cytosolic domain was expressed at a high level in a catalytically active form. The amount of soluble active enzyme in cell-free extracts of E. coli dramatically increased when the temperature during the induction was lowered from 37 degrees C to 22 degrees C.