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PubMed2001

Genes for the biosynthesis of spinosyns: applications for yield improvement in Saccharopolyspora spinosa.

Spinosyns A and D are the active ingredients in an insect control agent produced by fermentation of Saccharopolyspora spinosa. Spinosyns are macrolides with a 21-carbon, tetracyclic lactone backbone to which the deoxysugars forosamine and tri-O-methylrhamnose are attached. The spinosyn biosynthesis genes, except for the rhamnose genes, are located in a cluster that spans 74 kb of the S. spinosa genome. DNA sequence analysis, targeted gene disruptions and bioconversion studies identified five large genes encoding type I polyketide synthase subunits, and 14 genes involved in sugar biosynthesis, sugar attachment to the polyketide or cross-bridging of the polyketide. Four rhamnose biosynthetic genes, two of which are also necessary for forosamine biosynthesis, are located outside the spinosyn gene cluster. Duplication of the spinosyn genes linked to the polyketide synthase genes stimulated the final step in the biosynthesis--the conversion of the forosamine-less pseudoaglycones to endproducts. Duplication of genes involved in the early steps of deoxysugar biosynthesis increased spinosyn yield significantly.

Anti-Bacterial Agents↗
PubMed2006

The tallysomycin biosynthetic gene cluster from Streptoalloteichus hindustanus E465-94 ATCC 31158 unveiling new insights into the biosynthesis of the bleomycin family of antitumor antibiotics.

The tallysomycins (TLMs) belong to the bleomycin (BLM) family of antitumor antibiotics. The BLM biosynthetic gene cluster has been cloned and characterized previously from Streptomyces verticillus ATCC 15003, but engineering BLM biosynthesis for novel analogs has been hampered by the lack of a genetic system for S. verticillus. We now report the cloning and sequencing of the TLM biosynthetic gene cluster from Streptoalloteichus hindustanus E465-94 ATCC 31158 and the development of a genetic system for S. hindustanus, demonstrating the feasibility to manipulate TLM biosynthesis in S. hindustanus by gene inactivation and mutant complementation. Sequence analysis of the cloned 80.2 kb region revealed 40 open reading frames (ORFs), 30 of which were assigned to the TLM biosynthetic gene cluster. The TLM gene cluster consists of nonribosomal peptide synthetase (NRPS) genes encoding nine NRPS modules, a polyketide synthase (PKS) gene encoding one PKS module, genes encoding seven enzymes for deoxysugar biosynthesis and attachment, as well as genes encoding other biosynthesis, resistance, and regulatory proteins. The involvement of the cloned gene cluster in TLM biosynthesis was confirmed by inactivating the tlmE glycosyltransferase gene to generate a TLM non-producing mutant and by restoring TLM production to the DeltatlmE::ermE mutant strain upon expressing a functional copy of tlmE. The TLM gene cluster is highly homologous to the BLM cluster, with 25 of the 30 ORFs identified within the two clusters exhibiting striking similarities. The structural similarities and differences between TLM and BLM were reflected remarkably well by the genes and their organization in their respective biosynthetic gene clusters.

Amino Acid Sequence↗
PubMed2003

Inhibitor studies of isopentenyl pyrophosphate biosynthesis in suspension cultures of the yew Taxus chinensis var. mairei.

Some metabolic inhibitors, mevastatin (MVS), chlorocholine chloride (CCC), sodium pyrophosphate (NaPP) and D,L-glyceraldehyde (DLG), were respectively added into the suspension cultures of the yew Taxus chinensis var. mairei at the late phase of cell growth to study isopentenyl pyrophosphate (IPP) biosynthesis. The content of total taxanes decreased in the cases of MVS, NaPP and DLG addition, regardless of the inhibitor concentration, whereas it increased in the case of CCC addition. It was thus presumed that a mevalonate pathway might exist in IPP biosynthesis at the late growth phase of Taxus cells and that some IPP might transfer from the cytoplasm to the plastids. This presumption was also confirmed by analysing the effects of the inhibitors on taxane content. It was further demonstrated that IPP in the biosynthesis of taxol, baccatin III and 10-deacetylbaccatin III might transfer from the cytoplasm to the plastids, whereas the translocation of IPP might be not involved in cephalomannine biosynthesis. Furthermore, in combination with the results of previous studies, it is very probable that different IPP biosynthesis pathways exist during different growth phases in Taxus cells.

Bridged-Ring Compounds↗
PubMed1985

The effect of excess mevalonic acid on ubiquinone and tetrahymanol biosynthesis in Tetrahymena pyriformis.

When T. pyriformis is grown in the presence of 10(-2)M-mevalonic acid, the uptake exceeds the cell's requirement for this biosynthetic intermediate. The majority of the excess mevalonic acid is diverted into ubiquinone-8 biosynthesis whereas the biosynthesis of tetrahymanol, the major product of the mevalonic acid pathway, is unchanged. In the presence of excess external mevalonic acid, the biosynthesis of mevalonic acid by the cell is inhibited. It is proposed that ubiquinone biosynthesis is normally regulated by mevalonic acid availability, whereas tetrahymanol biosynthesis is regulated primarily at a later point in the pathway.

Mevalonic Acid↗
PubMed1990

Recycling of glucosylceramide and sphingosine for the biosynthesis of gangliosides and sphingomyelin in rat liver.

It was previously shown that sphingomyelin and gangliosides can be biosynthesized starting from sphingosine or sphingosine-containing fragments which originated in the course of GM1 ganglioside catabolism. In the present paper we investigated which fragments were specifically re-used for sphingomyelin and ganglioside biosynthesis in rat liver. At 30 h after intravenous injection of GM1 labelled at the level of the fatty acid ([stearoyl-14C]GM1) or of the sphingosine ([Sph-3H]) moiety, it was observed that radioactive sphingomyelin was formed almost exclusively after the sphingosine-labelled-GM1 administration. This permitted the recognition of sphingosine as the metabolite re-used for sphingomyelin biosynthesis. Conversely, gangliosides more complex than GM1 were similarly radiolabelled after the two treatments, thus ruling out sphingosine re-utilization for ganglioside biosynthesis. For the identification of the lipid fragment re-used for ganglioside biosynthesis, we administered to rats neutral glycosphingolipids (galactosylceramide, glucosylceramide and lactosylceramide) each radiolabelled in the sphingosine moiety or in the terminal sugar residue. Thereafter we compared the formation of radiolabelled gangliosides in the liver with respect to the species administered and the label location. After galactosylceramide was injected, no radiolabelled gangliosides were formed. After the administration of differently labelled glucosylceramide, radiolabelled gangliosides were formed, regardless of the position of the label. After lactosylceramide administration, the ganglioside fraction became more radioactive when the long-chain-base-labelled precursors were used. These results suggest that glucosylceramide, derived from glycosphingolipid and ganglioside catabolism, is recycled for ganglioside biosynthesis.

Animals↗
PubMed1991

Early steps of isoprenoid biosynthesis in Escherichia coli.

The incorporation of 2H- and 13C-labelled precursors into ubiquinone-8 (Uq-8) by strains of Escherichia coli was measured in order to define the pathway for the early steps in the biosynthesis of isoprenoids in these eubacteria. Cells grown with DL-[methyl-2H6]valine were found to label both the alpha-oxoisovaleric ('alpha-ketoisovaleric') acid alpha-oxoisohexanoic ('alpha-ketoisocaproic') acid, but not the Uq-8. Since these acids are required for the biosynthesis of isoprenoids by the acetolactate pathway, the operation of this pathway in the biosynthesis of Uq-8 is excluded. Cells grown with [1,2-13C2]acetate and non-labelled glucose readily incorporated 13C2 units into fatty acids, but failed to incorporate any label into the Uq-8. Cells grown with [U-13C6]glucose and non-labelled acetate, however, were found to label both the fatty acids and the Uq-8. Oxidative cleavage with periodate/permanganate of the Uq-8 isolated from cells grown with U-13C6-labelled glucose produced laevulinic acid, which was shown to be derived from two C2 units and one C1 unit of the labelled glucose by mass-spectral analysis of the 4,5-dihydro-6-methyl-2-phenylpyridazin-3(2H)-one derivative. The results of this work indicate that the C-2 and C-3 carbon unit of pyruvate, not acetyl-CoA, is the precursor to isopentenyl pyrophosphate (IPP) in these cells; however, the labelling pattern observed is consistent with the established acetoacetate pathway of isoprenoid biosynthesis. These data, coupled with the observed lack of inhibition of the growth of E. coli by mevinolin, a specific inhibitor of 3-hydroxy-3-methylglutaryl-CoA, can be best rationalized by the biosynthesis of IPP occurring in E. coli through a series of bound intermediates.

Acetates↗
PubMed1991

GTP-binding proteins may stimulate insulin biosynthesis in rat pancreatic islets by enhancing the signal-recognition-particle-dependent translocation of the insulin mRNA poly-/mono-some complex to the endoplasmic reticulum.

We aimed to elucidate the putative role of GTP-binding proteins in the regulation of insulin biosynthesis. For this purpose, freshly isolated rat islets were incubated in the presence of liposomes containing GDP, guanosine 5'-[beta-thio]diphosphate (GDP[S]), GTP, guanosine 5'-[gamma-thio]triphosphate (GTP[S]), guanosine 5'-[beta gamma-methylene]triphosphate (p[CH2]ppG), guanosine 5'[beta gamma-imido]triphosphate (p[NH]ppG) and ATP, and the effects of the liposomal delivery of these substances on rates of biosynthesis of insulin and total protein were determined. Insulin biosynthesis during a 1 h incubation at 1.67 mM-glucose was stimulated by ATP- and GTP[S]-containing liposomes as compared with control liposomes. At 16.7 mM-glucose, only the GTP[S]-containing liposomes stimulated insulin biosynthesis. No inhibition of islet protein and insulin synthesis was observed with GDP-, GDP[S]-, p[CH2]ppG- and p[NH]ppG-containing liposomes. By determining the subcellular distribution of insulin mRNA, it was found that the mRNA content associated with microsomes was increased and that associated with the cytosolic mono-/poly-somes decreased when the islets were incubated with GTP[S]-containing liposomes, resulting in an approximate doubling of the ratio of microsomal to polysomal-associated insulin mRNA. ATP-containing liposomes produced no effects on the association of insulin mRNA with microsomes. By using photoaffinity labelling and immunoprecipitation techniques, specific binding of GTP[35S] to the alpha-subunit of the signal-recognition particle (SRP) receptor in islet homogenates containing physiological concentrations of GTP and GDP was demonstrated. These findings suggest that the GTP-binding subunit(s) of the SRP receptor, and possibly also of other GTP-binding proteins involved in this process, may regulate insulin biosynthesis by stimulating the translocation of insulin mRNA to the endoplasmic reticulum and by increasing preproinsulin-peptide translocation into the lumen of the reticulum.

Adenosine Triphosphate↗
PubMed1993

Effects of fasting on phosphatidylcholine biosynthesis in hamster liver: regulation of cholinephosphotransferase activity by endogenous argininosuccinate.

The control of phosphatidylcholine biosynthesis in the hamster liver was examined. Livers of hamsters fasted for 24 and 48 h were perfused with labelled choline. Under both fasting conditions, the incorporation of labelled choline into phosphatidylcholine was reduced. After 48 h of fasting, the 52% reduction in phosphatidylcholine biosynthesis was caused by changes in several factors including a diminishing rate of choline uptake and severe reductions in the pool sizes of ATP and CTP (to 33-37% control values) which resulted in a decrease in the pools of choline-containing metabolites. The activation of cytidylyltransferase after 48 h of fasting might be regarded as a compensatory mechanism for the maintenance of phosphatidylcholine biosynthesis. After 24 h of fasting, a 25% reduction in phosphatidylcholine biosynthesis was observed. The ATP and CTP levels were decreased but the reduction was not severe enough to affect the choline uptake or the labelling of the phosphocholine fraction. The activities of the cytidylyltransferase remained unchanged but an accumulation of labelled CDP-choline was detected. Although choline-phosphotransferase activity was not changed in the microsomes, the enzyme activity was attenuated in the postmitochondrial fraction. Further analysis revealed that cholinephosphotransferase in the liver was inhibited by an endogenous inhibitor in the cytosol which was later identified as argininosuccinate. The level of argininosuccinate was elevated during fasting and the change quantitatively accounted for the attenuation of cholinephosphotransferase activity. The inhibition of choline-phosphotransferase by argininosuccinate, coupled with a substantial decrease in the diacylglycerol level, would provide the hamster liver with an immediate mechanism for the transient modulation of phosphatidylcholine biosynthesis during short-term fasting.

Animals↗
PubMed1993

Antagonism of phorbol-ester-stimulated phosphatidylcholine biosynthesis by the phospholipid analogue hexadecylphosphocholine.

The antagonization of phorbol 12-myristate 13-acetate (PMA)-stimulated phosphatidylcholine (PtdCho) biosynthesis by the phospholipid analogue hexadecylphosphocholine (HePC) in MDCK cells was investigated and compared with the corresponding influence in HeLa cells. In both cell lines, PMA-stimulated PtdCho biosynthesis was antagonized by 50 microM HePC. However, subsequent experiments provided evidence that PMA enhances PtdCho biosynthesis by at least two mechanisms: (i) by stimulation of choline uptake and (ii) by translocation of CTP:choline phosphate cytidylyltransferase to membranes. In MDCK cells, 5 nM PMA caused a 4-fold increase in [methyl-3H]choline incorporation into PtdCho, which was paralleled by an approx. 2-fold stimulation of choline uptake. These data indicate that choline uptake might play an important role in the regulation of PtdCho biosynthesis in this cell line, especially since we could not detect any significant increase in membrane-bound cytidyltransferase activity in PMA-treated MDCK cells. In contrast, enhanced PtdCho biosynthesis in HeLa cells is achieved by a 2-fold increase in particulate cytidylyltransferase activity after PMA stimulation. Translocation of cytidylyltransferase from the cytosol to membranes is therefore important in HeLa cells. Nevertheless, in both cell lines, the main target of HePC seems to be the translocation process. In MDCK cells, addition of 50 microM HePC decreases membrane-bound cytidylyltransferase activity by about 45%, compared with control cells and PMA-treated cells. In HeLa cells, PMA-induced translocation of cytidylyltransferase to membranes is totally abolished by HePC.

Animals↗
PubMed1994

Pteridine biosynthesis and nitric oxide synthase in Physarum polycephalum.

Physarum polycephalum, an acellular slime mould, serves as a model system to study cell-cycle-dependent events since nuclear division is naturally synchronous. This organism was shown to release isoxanthopterin which is structurally related to tetrahydrobiopterin, a cofactor of aromatic amino acid hydroxylases and of nitric oxide synthases (NOSs) (EC 1.14.13.39). Here, we studied Physarum pteridine biosynthesis in more detail and found that high amounts of tetrahydrobiopterin are produced and NOS activity is expressed. Physarum pteridine biosynthesis is peculiar in as much as 7,8-dihydroneopterin aldolase (EC 4.1.2.25), an enzyme of folic acid biosynthesis usually not found in organisms producing tetrahydrobiopterin, is detected in parallel. NOS purified from Physarum depends on NADPH, tetrahydrobiopterin and flavins. Enzyme activity is independent of exogenous Ca2+ and is inhibited by arginine analogues. The purified enzyme (with a molecular mass of 130 kDa) contains tightly bound tetrahydrobiopterin and flavins. During the synchronous cell cycle of Physarum, pteridine biosynthesis increases during S-phase whereas NOS activity peaks during mitosis, drops at telophase and peaks again during early S-phase. Our results characterize Physarum pteridine biosynthesis and NOS and suggest a possible link between NOS activity and mitosis.

Amino Acid Oxidoreductases↗
PubMed1995

Decrease in cardiac phosphatidylglycerol in streptozotocin-induced diabetic rats does not affect cardiolipin biosynthesis: evidence for distinct pools of phosphatidylglycerol in the heart.

Biosynthesis of phosphatidylglycerol (PG) and cardiolipin (CL) were investigated in perfused hearts of diabetic rats 4 days or 28 days after streptozotocin injection. Sham-injected and insulin-treated diabetic rats were used as controls. In addition, another group of rats fasted for 54 h was examined. Isolated rat hearts from these groups were perfused for 30 min with [32P]P(i), and the radioactivity incorporated into PG and CL and their pool sizes were determined in heart ventricles. There was no difference in the amount of radioactivity incorporated into CL, PG or other phospholipids between all groups. In addition, the pool sizes of CL and other phospholipids were unaltered. However, a striking decrease in the pool size of PG was observed in both diabetic and fasted rats compared to sham- and insulin-treated controls at 4 days after streptozotocin injection. The decrease in PG mass in diabetic rats was rapid (within 24-48 h) and was localized to cardiac membranes. Diabetes did not affect the activity of the enzymes of PG and CL biosynthesis in the mitochondrial fraction, or phospholipase A activity in subcellular fractions prepared from rat heart homogenates. In addition, pulse-chase experiments confirmed that diabetes did not affect the rate of new PG or CL biosynthesis. Since radioactivity associated with PG was unaltered in continuous-pulse perfusion experiments, a calculated 1.8-fold increase in the specific radioactivity of cardiac PG was observed in the hearts of acute diabetic rats compared with controls. Since the radioactivity incorporated into PG and CL, and the rate of CL biosynthesis, were unaltered in diabetic-rat hearts compared with controls, new CL was probably synthesized from newly synthesized PG. We postulate the existence of distinct pools of PG in the heart, and that the pool of newly synthesized PG used for CL biosynthesis does not appear to mix immediately with the pre-existing pool of PG in the isolated intact rat heart.

Animals↗
PubMed1995

The modulation of phosphatidylinositol biosynthesis in hamster hearts by methyl lidocaine.

Methyl lidocaine is an experimental anti-arrhythmic drug which has been shown to enhance the biosynthesis of phosphatidyl-inositol (PI) in the hamster heart. In this study, the effect of methyl lidocaine on enzymes involved in the biosynthesis of PI in the heart was examined. When the hamster heart was perfused with labelled methyl lidocaine, the majority of the compound was not metabolized after perfusion. The direct action of methyl lidocaine on an enzyme was studied by the presence of the drug in enzyme assays, whereas its indirect action was studied by assaying the enzyme activity in the heart after methyl lidocaine perfusion. CTP:phosphatidic acid cytidylyl-transferase, a rate-limiting enzyme in PI biosynthesis, was stimulated by methyl lidocaine in a direct manner. Kinetic studies revealed that methyl lidocaine caused a change in the affinity between the enzyme and phosphatidic acid and resulted in the enhancement of the reaction. Alternatively, acyl-CoA:lysophosphatidic acid acyltransferase, another key enzyme for PI biosynthesis, was not activated by the presence of methyl lidocaine. However, the enzyme activity was stimulated in hearts perfused with methyl lidocaine. The enhancement of the acyl-transferase by methyl lidocaine perfusion was found to be mediated via the adenylate cyclase cascade with the elevation of the cyclic AMP level. The stimulation of protein kinase A activity by cyclic AMP resulted in the phosphorylation and activation of the acyltransferase. Interestingly, the activity of protein kinase C was not stimulated by methyl lidocaine perfusion. We conclude that the enhancement of PI biosynthesis by methyl lidocaine in the hamster heart resulted from the direct activation of the cytidylyltransferase, as well as the phosphorylation and subsequent activation of the acyltransferase.

Acyltransferases↗
PubMed2000

Catecholamine biosynthesis and physiological regulation in neuroendocrine cells.

The catecholamines are widely distributed in mammals and their levels and physiological functions are regulated at many sites. These include their release from neuroendocrine cells, the type and sensitivity of the multiple receptors in target cells, the efficacy of the reuptake system in the secretory cells, and the rates of catecholamine biosynthesis and degradation. In the present review the main focus will be on the more recent studies on the biosynthesis in neuroendocrine cells which involves a specific set of enzymes, with special reference to physiologically important regulatory mechanisms. Eight enzymes of the biosynthetic pathway have now been identified, cloned, expressed as recombinant proteins, characterized with respect to catalytic and regulatory properties, and some of them also crystallized. The identification of the tyrosine hydroxylase catalysed reaction as the rate-limiting step in the normal catecholamine biosynthesis has attracted most attention, both in terms of transcriptional and post-translational regulation. In certain human genetic disorders of catecholamine biosynthesis other enzymes in the pathway may become rate-limiting, notably those involved in the biosynthesis/regeneration of the natural co-factor tetrahydrobiopterin in the tyrosine hydroxylase reaction. The enzymes involved seem to be regulated by a variety of physiological factors, both on a long-term scale and a short-term basis, and include the relative rates of synthesis, degradation and state of activation of the biosynthetic enzymes, notably of tyrosine hydroxylase. Multiple surface receptors and signalling pathways are activated in response to extracellular stimuli and play an essential role in the regulation of catecholamine biosynthesis.

Animals↗
PubMed2003

Steroid biosynthesis in prokaryotes: identification of myxobacterial steroids and cloning of the first bacterial 2,3(S)-oxidosqualene cyclase from the myxobacterium Stigmatella aurantiaca.

Steroids, such as cholesterol, are synthesized in almost all eukaryotic cells, which use these triterpenoid lipids to control the fluidity and flexibility of their cell membranes. Bacteria rarely synthesize such tetracyclic compounds but frequently replace them with a different class of triterpenoids, the pentacyclic hopanoids. The intriguing mechanisms involved in triterpene biosynthesis have attracted much attention, resulting in extensive studies of squalene-hopene cyclase in bacteria and (S)-2,3-oxidosqualene cyclases in eukarya. Nevertheless, almost nothing is known about steroid biosynthesis in bacteria. Only three steroid-synthesizing bacterial species have been identified before this study. Here, we report on a variety of sterol-producing myxobacteria. Stigmatella aurantiaca is shown to produce cycloartenol, the well-known first cyclization product of steroid biosynthesis in plants and algae. Additionally, we describe the cloning of the first bacterial steroid biosynthesis gene, cas, encoding the cycloartenol synthase (Cas) of S. aurantiaca. Mutants of cas generated via site-directed mutagenesis do not produce the compound. They show neither growth retardation in comparison with wild type nor any increase in ethanol sensitivity. The protein encoded by cas is most similar to the Cas proteins from several plant species, indicating a close evolutionary relationship between myxobacterial and eukaryotic steroid biosynthesis.

Amino Acid Sequence↗
PubMed2002

Interactions between sterol biosynthesis genes in embryonic development of Arabidopsis.

The sterol biosynthesis pathway of Arabidopsis produces a large set of structurally related phytosterols including sitosterol and campesterol, the latter being the precursor of the brassinosteroids (BRs). While BRs are implicated as phytohormones in post-embryonic growth, the functions of other types of steroid molecules are not clear. Characterization of the fackel (fk) mutants provided the first hint that sterols play a role in plant embryogenesis. FK encodes a sterol C-14 reductase that acts upstream of all known enzymatic steps corresponding to BR biosynthesis mutants. Here we report that genetic screens for fk-like seedling and embryonic phenotypes have identified two additional genes coding for sterol biosynthesis enzymes: CEPHALOPOD (CPH), a C-24 sterol methyl transferase, and HYDRA1 (HYD1), a sterol C-8,7 isomerase. We describe genetic interactions between cph, hyd1 and fk, and studies with 15-azasterol, an inhibitor of sterol C-14 reductase. Our experiments reveal that FK and HYD1 act sequentially, whereas CPH acts independently of these genes to produce essential sterols. Similar experiments indicate that the BR biosynthesis gene DWF1 acts independently of FK, whereas BR receptor gene BRI1 acts downstream of FK to promote post-embryonic growth. We found embryonic patterning defects in cph mutants and describe a GC-MS analysis of cph tissues which suggests that steroid molecules in addition to BRs play critical roles during plant embryogenesis. Taken together, our results imply that the sterol biosynthesis pathway is not a simple linear pathway but a complex network of enzymes that produce essential steroid molecules for plant growth and development.

Amino Acid Sequence↗
PubMed2001

Depolarization-stimulated catecholamine biosynthesis: involvement of protein kinases and tyrosine hydroxylase phosphorylation sites in situ.

Depolarizing stimuli increase catecholamine (CA) biosynthesis, tyrosine hydroxylase (TH) activity, and TH phosphorylation at Ser19, Ser31, and Ser40 in a Ca(2+)-dependent manner. However, the identities of the protein kinases that phosphorylate TH under depolarizing conditions are not known. Furthermore, although increases in Ser31 or Ser40 phosphorylation increase TH activity in vitro, the relative influence of phosphorylation at these sites on CA biosynthesis under depolarizing conditions is not known. We investigated the participation of extracellular signal-regulated protein kinase (ERK) and cAMP-dependent protein kinase (PKA) in elevated K(+)-stimulated TH phosphorylation in PC12 cells using an ERK pathway inhibitor, PD98059, and PKA-deficient PC12 cells (A126-B1). In the same paradigm, we measured CA biosynthesis. TH phosphorylation stoichiometry (PS) was determined by quantitative blot-immunolabeling using site- and phosphorylation state-specific antibodies. Treatment with elevated K(+) (+ 58 mM) for 5 min increased TH PS at each site in a Ca(2+)-dependent manner. Pretreatment with PD98059 prevented elevated K(+)-stimulated increases in ERK phosphorylation and Ser31 PS. In A126-B1 cells, Ser40 PS was not significantly increased by forskolin, and elevated K(+)-stimulated Ser40 PS was three- to five-fold less than that in PC12 cells. In both cell lines, CA biosynthesis was increased 1.5-fold after treatment with elevated K(+) and was prevented by pretreatment with PD98059. These results suggest that ERK phosphorylates TH at Ser31 and that PKA phosphorylates TH at Ser40 under depolarizing conditions. They also suggest that the increases in CA biosynthesis under depolarizing conditions are associated with the ERK-mediated increases in Ser31 PS.

Animals↗
PubMed1984

Effects of the aromatase inhibiting delta 1,4-bisnorcholadienic acid on the steroid biosynthesis in the human ovary in vitro.

Compounds capable of limiting oestrogen production may be useful for controlling fertility and growth of oestrogen-dependent tumors. To examine the direct effects of the aromatase inhibiting delta 1,4-bisnorcholadienic acid on the ovarian biosynthesis of steroids, tissue slices obtained from two cyclically functional human ovaries were incubated with [4-14C]pregnenolone as precursor. The steroid production was determined by measuring the concentration of eleven 14C-labelled steroids in the medium at the end of a 3-h incubation. In the ovary from the follicular phase the addition of delta 1,4-bisnorcholadienic acid to the incubation medium inhibited the biosynthesis of androstenedione, whereas the biosynthesis of 17 alpha-hydroxypregnenolone, dehydroepiandrosterone and androstenediol slightly increased. In the luteal ovary the in vitro-synthesis of progesterone, 17 alpha-hydroxyprogesterone, oestrone and oestradiol-17 beta was inhibited by delta 1,4-bisnorcholadienic acid. The principal manifestation of the effects of delta 1,4-bisnorcholadienic acid on the biosynthesis of steroids is an apparent inhibition of the 3 beta-hydroxysteroid dehydrogenase-delta 5-4-isomerase (3 beta-HSD) activity. The reduced production of oestrogens can be caused by an inhibition of the aromatase and/or the inhibition of the 3 beta-HSD activity. It is noteworthy, that the effects of the delta 1,4-bisnorcholadienic acid on the steroidogenesis in the human ovary are similar to those effects previously published for progestagens and the "pure antiandrogen" cyproterone. The results indicate, that the delta 1,4-bisnorcholadienic acid act directly on the steroid biosynthesis in the human ovary in vitro.

Aromatase Inhibitors↗
PubMed2006

Auxin biosynthesis in maize.

For the biosynthesis of the phytohormone indole-3-acetic acid (IAA), a number of tryptophan-dependent and -independent pathways have been discussed. Maize is an appropriate model system to analyze IAA biosynthesis particularly because high quantities of IAA conjugates are stored in the endosperm. This allowed precursor feeding experiments in a kernel culture system followed by retrobiosynthetic NMR analysis, which strongly suggested that tryptophan-dependent IAA synthesis is the predominant route for auxin biosynthesis in the maize kernel. Two nitrilases ZmNIT1 and ZmNIT2 are expressed in seeds. ZmNIT2 efficiently hydrolyzes indole-3-acetonitrile (IAN) to IAA and thus could be involved in auxin biosynthesis. Redundant pathways, e.g., via indole-3-acetaldehyde could imply that multiple mutants will be necessary to obtain IAA-deficient plants and to conclusively identify relevant genes for IAA biosynthesis.

Acetonitriles↗