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PubMed1968

Biosynthesis of phytoquinones. Biosynthetic origins of the nuclei and satellite methyl groups of plastoquinone, tocopherols and tocopherolquinones in maize shoots, bean shoots and ivy leaves.

1. By using dl-[ring-(14)C]phenylalanine, dl-[beta-(14)C]phenylalanine, dl-[alpha-(14)C]-tyrosine and dl-[beta-(14)C]tyrosine it was shown that in maize shoots (Zea mays) the nucleus and one nuclear methyl group of each of the following compounds, plastoquinone, gamma-tocopherol (aromatic nucleus) and alpha-tocopherolquinone, are formed from the nuclear carbon atoms and beta-carbon atom respectively of either exogenous phenylalanine or exogenous tyrosine. With ubiquinone only the aromatic ring of the amino acid is used in the synthesis of the quinone nucleus. Chemical degradation of plastoquinone and gamma-tocopherol molecules labelled from l-[U-(14)C]tyrosine established that a C(6)-C(1) unit directly derived from the amino acid is involved in the synthesis of these compounds. Radioactivity from [beta-(14)C]cinnamic acid is not incorporated into plastoquinone, tocopherols or tocopherolquinones, demonstrating that the C(6)-C(1) unit is not formed from any of the C(6)-C(1) phenolic acids associated with the metabolism of this compound. 2. The incorporation of radioactivity from l-[U-(14)C]tyrosine, dl-[beta-(14)C]tyrosine and dl-[U-(14)C]phenylalanine into bean shoots (Phaseolus vulgaris) and dl-[beta-(14)C]tyrosine and l-[Me-(14)C]methionine into ivy leaves (Hedera helix) was also investigated. Similar results were obtained to those reported for maize, except that in beans phenylalanine is only used for ubiquinone biosynthesis. This is attributed to the absence of phenylalanine hydroxylase from these tissues. In ivy leaves it is found that the beta-carbon atom of tyrosine gives rise to the 8-methyl group of delta-tocopherol, and it is suggested that for all other compounds examined it will give rise to the nuclear methyl group meta to the polyprenyl unit. 3. Preliminary investigations with the alga Euglena gracilis showed that in this organism ring-opening of tyrosine occurs to such an extent that the incorporation data from radiochemical experiments are meaningless. 4. The above results, coupled with previous observations, are interpreted as showing that in higher plants the nucleus of ubiquinone can be formed from either phenylalanine or tyrosine by a pathway involving as intermediates p-coumaric acid and p-hydroxybenzoic acid. Plastoquinone, tocopherols and alpha-tocopherolquinone are formed from p-hydroxyphenylpyruvate by a pathway in which the aromatic ring and C-3 of the side chain give rise respectively to the nucleus and to one nuclear methyl group. 5. Dilution experiments provided evidence that in maize shoots p-hydroxyphenylpyruvic acid and homogentisic acid (produced from p-hydroxyphenylpyruvic acid) are involved in plastoquinone biosynthesis, and presumably the biosynthesis of related compounds: however, other possible intermediates in the conversion including toluquinol (the aglycone of the proposed key intermediate) showed no dilution effects. Further, radioactivity from [Me-(14)C]toluquinol is not incorporated into any of the compounds examined. 6. Dilution experiments with 3,4-dihydroxybenzaldehyde and radioactive-labelling experiments with 3,4-dihydroxy[U-(14)C]benzoic acid demonstrated that these compounds are not involved in the biosynthesis of either ubiquinone or phylloquinone in maize shoots. 7. Evidence is also presented to show that in maize shoots ring-opening of the aromatic amino acids takes place. The suggestion is offered that this may take place via homogentisic acid, as in animals and some micro-organisms.

Amino Acids↗

PubMed1973

The effect of carbon monoxide on the nature of the accumulated 4,4-dimethyl sterol precursors of cholesterol during its biosynthesis from (2-14C)mevalonic acid in vitro.

Cholesterol biosynthesis was studied in rat liver subcellular fractions incubated with dl-[2-(14)C]mevalonic acid under gas phases consisting of either N(2)+O(2) (90:10) or CO+O(2) (90:10). CO inhibits cholesterol biosynthesis from [2-(14)C]mevalonic acid and results in a large accumulation of radioactive 4,4-dimethyl sterols. Separation of the components of the 4,4-dimethyl sterol fraction showed that lanosterol and dihydrolanosterol are the major components that accumulate during cholesterol biosynthesis in an atmosphere containing CO, whereas 14-demethyl-lanosterol and 14-demethyldihydrolanosterol are the major components of the much less intensely radioactive 4,4-dimethyl sterol fraction isolated from incubations with N(2)+O(2) as the gas phase. The identities of lanosterol, dihydrolanosterol and 14-demethyldihydrolanosterol were confirmed by both radiochemical and physicochemical methods, including g.l.c. and combined g.l.c.-mass spectrometry. CO therefore results in a qualitative as well as a quantitative difference in the 4,4-dimethyl sterol fraction which arises during cholesterol biosynthesis from mevalonic acid. The specific radioactivity of the [(14)C]lanosterol biosynthesized in the presence of CO was lower than that of its companion, [(14)C]dihydrolanosterol. The relative amounts of 4,4-dimethyl-Delta(24)-sterols and 4,4-dimethyl-24,25-dihydrosterols present in each type of incubation suggest that enzymic reduction of the sterol side chain occurs predominantly at a stage after that of lanosterol.

Animals↗

PubMed1976

The effect of N-acylglucosamines on the biosynthesis and secretion of insulin in the rat.

The effects of N-acylglucosamines on insulin release have been studied. N-Acylglucosamines stimulated insulin release from rat islets in vitro only if a sub-stimulatory concentration of glucose was also present, and this secretory response was abolished by mannoheptulose. In perifused islets the rapidity of the secreotry response to N-acetyl-D-glucosamine was similar to that observed with D-glucose. Increasing acyl-chain length from N-acetyl- to N-hexanoyl-D-glucosamine impaired the secretory response; however, N-dichloroacetyl-D-glucosamine was a more potent stimulator of release than was N-acetyl-D-glucosamine. Polymers of N-acetyl-D-glucosamine containing two to six monomers linked alpha1-4 did not stimulate insulin release; glucosamine linked to dextran via a propionyl or hexanoyl spacer group was also without insulin-releasing ability. N-Acylglucosamines were also effective in eliciting insulin release in vivo when injected into conscious rats. At the dose used (86 mumol), N-acetylgucosamine elicited a rapid rise in plasma-insulin concentration; N-butyrylglucosamine was less effective, and there was little or no response to N-hexanoylglucosamine. The response to N-dichloroacetyl-glucosamine was greater than that to N-acetylglucosamine; an increase in plasma insulin concentration could be elicited by N-dichloroacetylglucosamine at a dose (17 mumol) at which neither glucose nor N-acetylglucosamine was effective. The secretory response to acetylglucosamine is not mediated by conversion into glucose. Rates of (pro)-insulin biosynthesis by rat islets have been measured (Pro)-insulin biosynthesis was stimulated by glucose, and this response was abolished by mannoheptulose. N-Acetylglucosamine also stimulated (pro)-insulin biosynthesis; this effect of N-acetylglucosamine did not require the presence of glucose, and was not abolished by mannoheptulose. It is concluded that there are differences in signal reception and/or transduction for the processes of insulin biosynthesis and release.

Acetylglucosamine↗

PubMed1978

The influence of ammonia on purine and pyrimidine nucleotide biosynthesis in rat liver and brain in vitro.

1. The effect of ammonia on purine and pyrimidine nucleotide biosynthesis was studied in rat liver and brain in vitro. The incorporation of NaH(14)CO(3) into acid-soluble uridine nucleotide (UMP) in liver homogenates and minces was increased 2.5-4-fold on incubation with 10mm-NH(4)Cl plus N-acetyl-l-glutamate, but not with either compound alone. 2. The incorporation of NaH(14)CO(3) into orotic acid was increased 3-4-fold in liver homogenate with NH(4)Cl plus acetylglutamate. 3. The 5-phosphoribosyl 1-pyrophosphate content of liver homogenate was decreased by 50% after incubation for 10min with 10mm-NH(4)Cl plus acetylglutamate. 4. Concomitant with this decrease in free phosphoribosyl pyrophosphate was a 40-50% decrease in the rates of purine nucleotide synthesis, both de novo and from the preformed base. 5. Subcellular fractionation of liver indicated that the effects of NH(4)Cl plus acetylglutamate on pyrimidine and purine biosynthesis required a mitochondrial fraction. This effect of NH(4)Cl plus acetylglutamate could be duplicated in a mitochondria-free liver fraction with carbamoyl phosphate. 6. A similar series of experiments carried out with rat brain demonstrated a significant, though considerably smaller, effect on UMP synthesis de novo and purine base reutilization. 7. These data indicate that excessive amounts of ammonia may interfere with purine nucleotide biosynthesis by stimulating production of carbamoyl phosphate through the mitochondrial synthetase, with the excess carbamoyl phosphate in turn increasing pyrimidine nucleotide synthesis de novo and diminishing the phosphoribosyl pyrophosphate available for purine biosynthesis.

Ammonia↗

PubMed1981

Effect of diethylstilboestrol on phosphatidylcholine biosynthesis and choline metabolism in the liver of roosters.

It has been known for 40 years that oestrogens stimulate phospholipid metabolism in roosters. We have investigated in vivo the mechanism for this effect. Young roosters were injected daily with 1 mg of diethylstilboestrol for 1--3 days. At 4 h after the last injection, 30 microCi of [Me-3H]choline was injected into the portal vein. At periods up to 3 min the livers were freeze-clamped and choline and its metabolites were extracted and resolved by t.l.c. Hormone treatment in the first 2 days resulted in a 2-fold increase in phosphorylation of [Me-3H]choline and a decrease in the oxidation of [Me-3H]choline to [3H]betaine. The concentrations of phosphocholine in liver were increased 2-fold during the first 2 days concomitant with a 2-fold increase in the rate of phosphatidylcholine biosynthesis. After 3 days of hormone treatment, many of the above effects were reversed and the rate of phosphatidylcholine biosynthesis decreased to approx. 60% of the control value. The results suggest that the initial hormone treatments activate choline kinase within 4 h and, thereby, divert choline form oxidation to betaine. The resulting increased phosphocholine concentrations cause an increase in the activity of CTP:phosphocholine cytidylyltransferase, which results in a doubling of the rate of phosphatidylcholine biosynthesis. After 3 days of hormone treatment, the biosynthesis of phosphatidylcholine is decreased, most likely by an effect on the cytidylyltransferase reaction.

Animals↗

PubMed2003

Overexpression of UDP-glucose dehydrogenase in Escherichia coli results in decreased biosynthesis of K5 polysaccharide.

The Escherichia coli K5 capsular polysaccharide (glycosaminoglycan) chains are composed of the repeated disaccharide structure: -GlcAbeta1,4-GlcNAcalpha1,4-(where GlcA is glucuronic acid and GlcNAc is N-acetyl-D-glucosamine). The GlcA, present in most glycosaminoglycans, is donated from UDP-GlcA, which, in turn, is generated from UDP-glucose by the enzyme UDP-glucose dehydrogenase (UDPGDH). The formation of UDP-GlcA is critical for the biosynthesis of glycosaminoglycans. To investigate the role of UDPGDH in glycosaminoglycan biosynthesis, we used K5 polysaccharide biosynthesis as a model. E. coli was transformed with the complete gene cluster for K5 polysaccharide production. Additional transformation with an extra copy of UDPGDH resulted in an approx. 15-fold increase in the in vitro UDPGDH enzyme activity compared with the strain lacking extra UDPGDH. UDP-GlcA levels were increased 3-fold in overexpressing strains. However, metabolic labelling with [14C]glucose showed, unexpectedly, that overexpression of UDPGDH lead to decreased formation of K5 polysaccharide. No significant difference in the K5 polysaccharide chain length was observed between control and overexpressing strains, indicating that the decrease in K5-polysaccharide production most probably was due to synthesis of fewer chains. Our results suggest that K5-polysaccharide biosynthesis is strictly regulated such that increasing the amount of available UDP-GlcA results in diminished K5-polysaccharide production.

Bacterial Capsules↗

PubMed2007

Phospholipid scramblase-3 regulates cardiolipin de novo biosynthesis and its resynthesis in growing HeLa cells.

PLS3 (phospholipid scramblase-3) is a new member of the family of phospholipid scramblases and transports CL (cardiolipin) from the inner to the outer mitochondrial membrane. In the present paper we examined whether changing the levels of functional PLS3 in HeLa cells altered de novo CL biosynthesis and its resynthesis. HeLa cells overexpressing PLS3 or expressing a disrupted PLS3 (F258V) or control were incubated with [1,3-3H]glycerol and radioactivity incorporated into CL was determined. CL biosynthesis from [1,3-3H]glycerol was increased 1.8-fold in PLS3 cells and 2.1-fold in F258V cells compared with control. This was due to a 64% (P<0.05) and 2.6-fold (P<0.05) elevation in CL synthase activity in PLS3 and F258V cells respectively, compared with control, and not due to changes in phosphatidylglycerolphosphate synthase activity. The increase in CL synthase activity in these cells was due to an increase in its mRNA expression. In contrast, resynthesis of CL from [1-14C]linoleic acid was reduced 52% (P<0.05) in PLS3 and 45% (P<0.05) in F258V cells compared with control and this was due to a reduction in mitochondrial monolysocardiolipin acyltransferase activity. Although protein levels of mitochondrial monolysocardiolipin acyltransferase were unaltered, activity and mRNA expression of endoplasmic reticulum monolysocardiolipin acyltransferase was upregulated in PLS3 and F258V cells compared with controls. These data indicate that the CL resynthesis in HeLa cells is sensitive to the mitochondrial localization of CL and not the level of the reacylating enzymes. Alterations in functional PLS3 levels in PLS3 or F258V cells did not affect the mitochondrial decarboxylation of phosphatidylserine to phosphatidylethanolamine indicating that the biosynthetic changes to CL were specific for this mitochondrial phospholipid. We hypothesize that the cardiolipin resynthesis machinery in the cell 'senses' altered levels of CL on mitochondrial membranes and that de novo CL biosynthesis is up-regulated in HeLa cells as a compensatory mechanism in response to altered movement of mitochondrial CL. The results identify PLS3 as a novel regulator of CL de novo biosynthesis and its resynthesis.

Blotting, Western↗

PubMed1994

Cardiolipin biosynthesis in the isolated heart.

The pathway for the biosynthesis of new cardiolipin was investigated in the isolated perfused intact rat heart. Isolated rat hearts were perfused in the Langendorff mode for up to 60 min with Krebs-Henseleit buffer containing 0.1 microM [U-14C]glycerol. Analysis of radioactivity incorporated into phospholipids in the organic phase revealed an increase in radioactivity incorporated into phosphatidylglycerol, cardiolipin and other phospholipids with time of perfusion. This was associated with a loss of radioactivity from phosphatidic acid. In contrast, perfusion of hearts for up to 60 min with 0.1 mM [1,(3)-3H]glycerol in the perfusate revealed an increased radioactivity associated with phosphatidic acid as well as cardiolipin, phosphatidylglycerol and other phospholipids. Perfusion of hearts for up to 60 min with [32P]Pi in the perfusate revealed a time-dependent increase in radioactivity associated with all phospholipids. Perfusion of hearts for up to 60 min with 0.1 microM or 0.1 mM glycerol in the perfusate did not affect the concentration of phosphatidic acid, cardiolipin or phosphatidylglycerol. To determine the rate-limiting step of cardiolipin biosynthesis, hearts were pulsed for 5 min with 0.1 microM [1,(3)-3H]glycerol and chased for up to 60 min with 0.1 microM glycerol in the perfusate. Radioactivity was maximum at the start of the chase in phosphatidic acid (and 1,2-diacylglycerol), and was subsequently chased into phosphatidylinositol, phosphatidylglycerol and cardiolipin (and other phospholipids). Significant radioactivity in phosphatidylglycerol phosphate was not detected. Radioactivity in CDP-sn-1,2-diacylglycerol remained constant throughout the chase. The activities of the enzymes of the Kennedy pathway for cardiolipin biosynthesis in the heart were determined. On the basis of continuous-pulse and pulse-chase labelling studies it is postulated that the cardiac polyglycerophospholipids phosphatidylglycerol and cardiolipin are actively synthesized from newly synthesized phosphatidic acid via the Kennedy pathway. In addition, the results suggest that the rate-limiting step of cardiolipin biosynthesis in the intact heart is probably the conversion of phosphatidic acid into CDP-sn-1,2-diacylglycerol.

Animals↗

PubMed1994

Plasmenylcholine (1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphocholine) biosynthesis in guinea-pig heart and liver: cholinephosphotransferase is a bifunctional enzyme for the synthesis of phosphatidylcholine and plasmenylcholine.

Plasmenylcholine is present in significant proportion (32% of choline phosphoglycerides) in the guinea-pig heart but exists as a minor component (3% of choline phosphoglycerides) in the guinea-pig liver. In this study, the biosynthesis of plasmenylcholine in these two organs was examined. The organs were perfused with labelled choline for 15 min and chased with unlabelled choline for up to 7 h. The labelling of phosphatidylcholine was 6-fold higher than that of plasmenylcholine in the heart and about 60-fold higher in the liver. However, the same labelling ratio was maintained throughout the chase period in both organs. Alterations in the specific radioactivity of CDP-choline caused corresponding changes in the labelling of phosphatidylcholine and plasmenylcholine. Our results suggest that in guinea-pig heart and liver, CDP-choline is the immediate precursor of biosynthesis of phosphatidylcholine and plasmenylcholine. The biochemical cause for the difference in their rates of formation between the two organs was explored. The enzyme activities for the formation of both choline phosphoglycerides were determined. The two reactions share the same characteristics, and 1,2-diacylglycerol and 1-alk-1'-enyl-2-acylglcerol were found to be mutually inhibitory in a competitive fashion. The pool sizes of 1,2-diacylglycerol and 1-alk-1'-enyl-2-acylglycerol were determined, and their ratios were found to be 42 in the heart and 422 in the liver. We conclude that cholinephosphotransferase catalyses the formation of both phosphatidylcholine and plasmenylcholine in the guinea-pig tissues and the rate of plasmenylcholine biosynthesis is dependent on the availability of 1-alk-1'-enyl-2-acylglycerol. Plasmenylcholine biosynthesis is also subjected to modulation by the 1,2-diacylglycerol content of the tissue.

Animals↗

PubMed1996

Isolation and characterization of a Chinese hamster ovary (CHO) mutant defective in the second step of glycosylphosphatidylinositol biosynthesis.

Mutant cell lines defective in the biosynthesis of glycosylphosphatidylinositol (GPI) described to date were isolated by selecting cells which no longer expressed one or more endogenous GPI-anchored proteins on their surface. In this study, a new mutant in this pathway was isolated from ethylmethane-sulphonate-mutagenized Chinese hamster ovary cells stably transfected with human placental alkaline phosphatase (PLAP) as a marker of GPI-anchored proteins. A three-step protocol was employed. In the first step, cells with decreased surface expression of PLAP were selected by four rounds of complement-mediated lysis with an anti-(alkaline phosphatase) antibody. The surviving cells were cloned by limiting dilution and those with low levels of total alkaline phosphatase activity were selected in the second step. Finally, the ability of each clone to synthesize the first three intermediates in GPI biosynthesis in vitro was assessed to determine which cells with low alkaline phosphatase activity harboured a defect in one of these reactions. Of 230 potential mutants, one was defective in the second step of GPI biosynthesis. Microsomes from this mutant, designated G9PLAP.85, were completely unable to deacetylate either endogenous GlcNAc-phosphatidylinositol (PI) synthesized from UDP[6-3H]GlcNAc or exogenous GlcNAc-PI added directly to the membranes. Complementation analysis with the Thy-1-deficient murine lymphoma cells demonstrated that G9PLAP.85 has a molecular defect distinct from these previously described mutants. Therefore, these results suggest that mutants in GPI biosynthesis could be selected from almost any cell line expressing a GPI-anchored marker protein.

Acetylglucosamine↗

PubMed1996

Determination of the primary target for isoniazid in mycobacterial mycolic acid biosynthesis with Mycobacterium aurum A+.

The target of the potent antituberculosis drug isoniazid was investigated in Mycobacterium aurum A+, against which isoniazid has an MIC (the minimum concentration required to give growth inhibition) of 0.3 microgram/ml. Mycolic acid biosynthesis, measured by the incorporation of label from [1-14C]acetate into mycolic acids, was inhibited differentially by isoniazid in cell-wall preparations of M. aurum A+. Thus at an isoniazid concentration of 1 microgram/ml, mycolic acid biosynthesis was inhibited by 80% but concomitant biosynthesis of non-hydroxylated fatty acids was inhibited by only 15%. Three lines of evidence identified 24:1 cis-5 elongase as the primary isoniazid target. First, 24:1 cis-5 did not restore isoniazid-inhibited mycolic acid biosynthetic activity in a crude cell-wall preparation, suggesting that the drug acts after the formation of the delta-5 double bond. Secondly, a 24:1 cis-5 elongase assay in which the product is mycolic acid is completely inhibited by isoniazid. Finally, the only intermediates that accumulate as a result of the addition of isoniazid are acids of 24 carbons. Both 24:0 and 24:1 are observed in a similar ratio whether or not isoniazid is present, even though concomitant mycolic acid biosynthesis is inhibited by isoniazid. These results are consistent with studies of the M. tuberculosis InhA protein by Dessen, Quemard, Blanchard, Jacobs and Sacchettini [(1995) Science 267, 1638-1641].

Acetyltransferases↗

PubMed1997

Biosynthesis of the labdane diterpene marrubiin in Marrubium vulgare via a non-mevalonate pathway.

The biosynthesis of the furanic labdane diterpene marrubiin has been studied in plantlets and shoot cultures of Marrubium vulgare (Lamiaceae). The use of [2-14C]acetate, [2-14C]pyruvate, [2-14C]mevalonic acid and [U-14C]glucose incorporation experiments showed that the labelling of sterols in etiolated shoot cultures of M. vulgare was in accordance with their biosynthesis via the acetate-mevalonate pathway. In contrast, the incorporation rates of these precursors into the diterpene marrubiin could not be explained by biosynthesis of this compound via the acetate-mevalonate pathway. Cultivation of etiolated shoot cultures of M. vulgare on medium containing [1-13C]glucose and subsequent 13C-NMR spectroscopy of marrubiin led to the conclusion that the biosynthesis of marrubiin follows a non-mevalonate pathway. All isoprenic units of 13C-labelled marrubiin were enriched in those carbons that correspond to positions 1 and 5 of a putative precursor isopentenyl diphosphate. This labelling pattern from [1-13C]glucose is consistent with an alternative pathway via trioses, which has already been shown to occur in Eubacteria and Gymnospermae. The labdane skeleton is a precursor of many other skeletal types of diterpenes. Therefore it becomes obvious that in connection with the few known examples of a non-mevalonate pathway to isoprenoids the formation of some isoprenoids in plants via a non-mevalonate pathway might be quite common.

Acetates↗

PubMed1998

Neuropeptide regulation of biosynthesis of the juvenoid, methyl farnesoate, in the edible crab, Cancer pagurus.

The neuropeptide mandibular organ (MO)-inhibiting hormone (MO-IH), synthesized and secreted from the X-organ-sinus-gland complex of the eyestalk, regulates the biosynthesis of the putative crustacean juvenile hormone, methyl farnesoate (MF). Using radiolabelled acetate as a precursor for isoprenoid biosynthesis, farnesoic acid (FA), farnesol, farnesal, MF and geranyl geraniol were detected in MOs cultured for 24 h. Treatment of MOs with extract of sinus gland inhibited the final step of biosynthesis of MF, catalysed by FA O-methyltransferase. Additionally, treatment of MOs with purified MO-IH exhibited a dose-dependent inhibition of this final step of MF synthesis. The extent of this inhibition was dependent on the ovary stage of the MO-donor animal, being maximal in MOs from animals in the early stages of ovarian development. Assay of FA O-methyltransferase activity, using [3H]FA in the presence of S-adenosyl-l-methionine, demonstrated that the enzyme was located in the cytosolic fraction of MOs and was inhibited by incubation of MOs with MO-IH prior to preparation of subcellular fractions. For cytosolic preparations taken from vitellogenic animals, both Vmax and Km were appreciably lower than for those taken from non-vitellogenic animals. Conversely, eyestalk ablation of early-vitellogenic animals, which removes the source of MO-IH in vivo, resulted in enhancement of the cytosolic FA O-methyltransferase activity. Although both Vmax and Km show an appreciable increase upon eyestalk ablation, the increased enzyme activity is probably reflected by the fact that Vmax/Km (an approximate indication of kcat) has increased 5-fold. The combined evidence demonstrates that MO-IH inhibits FA O-methyltransferase, the enzyme which catalyses the final step of MF biosynthesis in MOs.

Animals↗

PubMed2002

Identification of members of the Aspergillus nidulans SREA regulon: genes involved in siderophore biosynthesis and utilization.

Iron is an essential trace element for almost all organisms. However, an excess of this metal within cells can be deleterious on account of catalysis of cell-damaging hydroxyl radicals. Therefore, the concentration of iron within cells is tightly regulated and the primary control occurs by regulating its uptake. Under conditions of low iron availability, most fungi mobilize extracellular iron by excretion of low-molecular-mass ferric iron chelators, termed siderophores. Due to the potential impact of iron metabolism on fungal pathogenicity, a better insight into siderophore-mediated iron uptake is needed. In Aspergillus nidulans, siderophore biosynthesis and uptake are negatively regulated by the GATA-type transcription factor SREA. Hence, genes involved in siderophore biosynthesis and uptake are characterized by transcriptional induction under iron limitation in wild-type strain and de-repression in an sreA-deletion strain under conditions of sufficient iron supply. Such genes have been searched for using different strategies, e.g. differential mRNA display and expression analysis of candidate genes from various A. nidulans sequence databases. The identified genes presumably encode enzymes needed for siderophore biosynthesis, and transporters involved in siderophore uptake and/or excretion. The functional characterization of these genes will help to unravel the pathways involved in siderophore biosynthesis and uptake.

Aspergillus nidulans↗

PubMed1999

Counter-regulatory effect of sodium butyrate on tumour necrosis factor-alpha (TNF-alpha)-induced complement C3 and factor B biosynthesis in human intestinal epithelial cells.

The various biological activities of butyrate have been well documented. In this study, we tested the effects of butyrate on TNF-alpha-induced complement C3 and factor B biosynthesis in human intestinal epithelial cells. The biosynthesis of C3, factor B and IL-8 was evaluated at the protein and mRNA levels. To evaluate transcriptional activation, the nuclear run-on assay was performed. The transcription factor-DNA binding activity was assessed by an electrophoretic gel mobility shift assay (EMSA). In the intestinal epithelial cell lines HT-29, T84 and Caco-2, sodium butyrate enhanced TNF-alpha-induced C3 secretion, but suppressed TNF-alpha-induced factor B and IL-8 secretion. Nuclear run-on assay revealed that transcriptional regulatory mechanisms are involved in the effects of sodium butyrate. The EMSAs indicated that sodium butyrate suppressed TNF-alpha-induced nuclear factor (NF)-kappaB- and activation protein (AP)-1-DNA binding activity, but enhanced TNF-alpha-induced activation of CCAAT/enhancer-binding protein (C/EBP)beta (NF-IL-6)-DNA binding activity. Sodium butyrate induced a counter-regulatory effect on TNF-alpha-induced C3 and factor B biosynthesis in human intestinal epithelial cells. Butyrate action has been discussed with its activity to induce histone hyperacetylation, but its counter-regulatory effect on complement biosynthesis may be closely associated with the modulation of transcription factor activation.

Blotting, Northern↗

PubMed2001

Exopolysaccharide (EPS) biosynthesis by Lactobacillus sakei 0-1: production kinetics, enzyme activities and EPS yields.

AIMS: To determine optimal exopolysaccharide (EPS) production conditions of the mesophilic lactic acid bacterium strain Lactobacillus sakei 0-1 and to detect possible links between EPS yields and the activity of relevant enzymes. METHODS AND RESULTS: Fermentation experiments at different temperatures using either glucose or lactose were carried out. EPS production took place during the exponential growth phase. Low temperatures, applying glucose as carbohydrate source, resulted in the best bacterial growth, the highest amounts of EPS and the highest specific EPS production. Activities of 10 important enzymes involved in the EPS biosynthesis and the energy formation of Lact. sakei 0-1 were measured. The obtained results revealed that there is a clear link for some enzymes with EPS biosynthesis. It was also demonstrated clearly that the presence of rhamnose in the EPS building blocks is due to high activities of the enzymes involved in the rhamnose synthetic branch. CONCLUSION: EPS production in Lact. sakei 0-1 is growth-associated and displays primary metabolite kinetics. Glucose as carbohydrate source and low temperatures enhance the EPS production. The enzymes involved in the biosynthesis of the activated sugar nucleotides play a major role in determining the monomeric composition of the synthesized EPS. SIGNIFICANCE AND IMPACT OF THE STUDY: The proposed results contribute to a better understanding of the physiological factors influencing EPS production and the key enzymes involved in EPS biosynthesis by Lact. sakei.

Bacterial Capsules↗

PubMed1999

Ontogeny and sexual differentiation of somatostatin biosynthesis and secretion in the hypothalamic periventricular-median eminence pathway.

The biosynthesis of somatostatin (SRIH) in the hypothalamic periventricular nucleus (PeN) is sexually differentiated in neonatal and adult rats by virtue of the organizational and activational actions, respectively, of sex steroid hormones. Little information exists, however, on the normal pattern of maturation of these neurones or on how the sexually differentiated biosynthesis may relate to ontogenetic changes in somatostatin secretion during the neonatal and pubertal periods of development. Hence in the present study we determined the postnatal developmental profile of SRIH mRNA and peptide levels in the PeN-median eminence (ME) pathway as well as SRIH secretion, using an acute explant preparation, from the day of birth, through puberty and into adulthood in male and female rats. The results demonstrate that: (1) developmental sex differences in SRIH biosynthesis in PeN neurones occurred in an orderly cascade with differences observed for mRNA expression at postnatal day 5, for peptide content in the perikarya at postnatal day 10 and for peptide content in the nerve terminal (ME) by postnatal day 25; (2) sex differences in SRIH release were not evident prior to postnatal day 40; and (3) the developmental profile of SRIH biosynthesis in PeN neurones is unique compared with other hypothalamic (ventromedial nucleus) and extrahypothalamic (parietal cortex) populations. Specific developmental changes in the biosynthetic and secretory activity of the hypothalamic SRIH PeN-ME pathway may have a functional importance in the maturation of hypothalamic SRIH pathways involved in the regulation of GH secretion.

Aging↗

PubMed1997

The LS locus of pea encodes the gibberellin biosynthesis enzyme ent-kaurene synthase A.

Gibberellins (GAs) are hormones required for several aspects of plant development, including internode elongation and seed development in pea (Pisum sativum L.). The first committed step in the GA biosynthesis pathway is the conversion of geranylgeranyl diphosphate (GGDP) to ent-kaurene via copalyl diphosphate (CDP). These two reactions are catalyzed by the cyclases ent-kaurene synthase A (KSA) and ent-kaurene synthase B (KSB), respectively. Previous genetic and biochemical analysis of the GA-responsive ls-1 mutant of pea suggested that GA levels are reduced in a developmental- and organ-specific manner due to reduced GA biosynthesis. Analysis of cellfree enzyme preparations from WT and ls-1 embryos at contact point reveals that ls-1 reduces the activity of KSA but not KSB. To characterize the ls-1 mutation in more detail, a cDNA coding for a pea KSA was cloned and shown to be encoded by the LS locus. The ls-1 mutation results from an intronic G to A substitution that causes impaired RNA splicing. To determine the activity of the KSAs encoded by the LS and ls-1 alleles, a new in vitro assay for combined KSA and KSB activity has been developed using the KSB gene of pumpkin. Using recombinant WT KSA and KSB fusion proteins, GGDP is converted to ent-kaurene in vitro. Based on the sequence of RT-PCR products, three different truncated KSA proteins are predicted to exist in ls-1 plants. The most abundant mutant KSA protein does not possess detectable activity in vitro. Nevertheless, the ls-1 allele is not null and is able to encode at least a partially functional KSA since a more severe is allele has been identified. The ls-1 mutation has played a key role in identifying a role for GAs in pea seed development in the first few days after fertilization, but not in older seeds. KSA expression in seeds is developmentally regulated and parallels overall GA biosynthesis, suggesting that KSA expression may play an important role in the regulation of GA biosynthesis and seed development.

Alkyl and Aryl Transferases↗