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PubMed1980

The biosynthesis of cyanogenic glucosides in higher plants. Channeling of intermediates in dhurrin biosynthesis by a microsomal system from Sorghum bicolor (linn) Moench.

The biosynthetic pathway for the cyanogenic glucoside, dhurrin, involves the following intermediates: L-tyrosine, N-hydroxytyrosine, p-hydroxyphenylacetaldoxime, p-hydroxyphenylacetonitrile, and p-hydroxymandelonitrile. N-Hydroxytyrosine and p-hydroxy-phenylacetonitrile produced from L-tyrosine by microsomes from seedlings of Sorghum bicolor are utilized more effectively as substrates than exogenously added N-hydroxytyrosine and p-hydroxyphenylacetonitrile. The minimum values for the channeling ratios are 25 for N-hydroxytyrosine and 115 for p-hydroxyphenylacetonitrile. On the other hand, p-hydroxyphenylacetaldoxime produced internally exchanges readily with exogenously added p-hydroxyphenylacetaldoxime. These results indicate that the biosynthetic pathway is catalyzed by two mutienzyme complexes or by two multifunctional proteins and explain why the rate of the overall sequential reaction starting from L-tyrosine is greater than the rates of reaction initiated later in the sequence with the known intermediates N-hydroxytyrosine and p-hydroxyphenylacetonitrile. Attempts to cross-link chemically the last enzyme in the pathway, a soluble UDP-glucose glucosyl-transferase, to the microsomal system were unsuccesful.

Carbon Radioisotopes↗
PubMed1981

[Activities of 3-hydroxyl-3-methylglutaryl-CoA reductase and acetyl-CoA carboxylase and the rate of mevalonic acid, squalene, sterol and fatty acid biosynthesis from [1-14C]acetyl-CoA and [2-14C]malonyl-CoA in rat liver: effects of Triton WR 1339, starvation and cholesterol diet].

The effects of Triton WR 1339, starvation and cholesterol diet on the activities of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) and acetyl-CoA carboxylase and on the rates of mevalonic acid (MVA) biosynthesis from acetyl-CoA and malonyl-CoA in the soluble (140 000 g) and microsomal fractions of rat liver, on the rate of incorporation of these substrates into squalene, cholesterol and lanosterol in the rat liver postmitochondrial fraction and on the rate of fatty acid biosynthesis was studied. The administration of Triton WR 1339 (200 mg per 100 g of body weight twice) stimulated the activity of HMG-CoA reductase and MVA biosynthesis from acetyl-CoA and malonyl-CoA in the intact and solubilized microsomal fractions and had no effect on these parameters in the soluble fraction. Starvation for 36 hrs did not cause inhibition of the reductase activity or MVA biosynthesis from both substrates in the soluble fraction. Alimentary cholesterol significantly increased the activity of HMG-CoA reductase, had no effect on the rate of MVA biosynthesis from acetyl-CoA and stimulated the malonyl-CoA incorporation in to MVA in the soluble fraction. Starvation an alimentary cholesterol inhibited the HMG-CoA reductase activity and MVA biosynthesis from both substrates in the solubilized microsomal fraction. Triton WR 1339 stimulated 4--19-fold the lipid formation in the total unsaponified fraction and its components i.e. squalene, lanosterol, cholesterol, from acetyl-CoA and only insignificantly (1,2--1,7-fold) increased malonyl-CoA incorporation into these compounds. Starvation and alimentary cholesterol repressed lanosterol and cholesterol biosynthesis from acetyl-CoA, decreased malonyl-CoA incorporation into these sterols and had no influence on squalene biosynthesis from the two substrates. Triton WR 1339 and starvation inhibited the acetyl-CoA carboxylase activity, unaffected by alimentary cholesterol. No significant changes in the rate of fatty acid biosynthesis from the substrates were observed. The data obtained provide evidence for the existence of autonomic pathways of MVA biosynthesis localized in the soluble and microsomal fractions of rat liver. The pathway of MVA biosynthesis in the soluble fraction is less sensitive to regulatory factors. Sterol biosynthesis from malonyl-CoA is also more resistant to regulatory effects than sterol biosynthesis from acetyl-CoA. This suggests that HMG-CoA reductase localized in the soluble fraction takes part in MVA and sterol biosynthesis from malonyl-CoA.

Acetyl Coenzyme A↗
PubMed2006

Nitric oxide mediates the fungal elicitor-induced puerarin biosynthesis in Pueraria thomsonii Benth. suspension cells through a salicylic acid (SA)-dependent and a jasmonic acid (JA)-dependent signal pathway.

Nitric oxide (NO) has emerged as a key signaling molecule in plant secondary metabolite biosynthesis recently. In order to investigate the molecular basis of NO signaling in elicitor-induced secondary metabolite biosynthesis of plant cells, we determined the contents of NO, salicylic acid (SA), jasmonic acid (JA), and puerarin in Pueraria thomsonii Benth. suspension cells treated with the elicitors prepared from cell walls of Penicillium citrinum. The results showed that the fungal elicitor induced NO burst, SA accumulation and puerarin production of P. thomsonii Benth. cells. The elicitor-induced SA accumulation and puerarin production was suppressed by nitric oxide specific scavenger cPITO, indicating that NO was essential for elicitor-induced SA and puerarin biosynthesis in P. thomsonii Benth. cells. In transgenic NahG P. thomsonii Benth. cells, the fungal elicitor also induced puerarin biosynthesis, NO burst, and JA accumulation, though the SA biosynthesis was impaired. The elicitor-induced JA accumulation in transgenic cells was blocked by cPITO, which suggested that JA acted downstream of NO and its biosynthesis was controlled by NO. External application of NO via its donor sodium nitroprusside (SNP) enhanced puerarin biosynthesis in transgenic NahG P. thomsonii Benth. cells, and the NO-triggered puerarin biosynthesis was suppressed by JA inhibitors IBU and NDGA, which indicated that NO induced puerarin production through a JA-dependent signal pathway in the transgenic cells. Exogenous application of SA suppressed the elicitor-induced JA biosynthesis and reversed the inhibition of IBU and NDGA on elicitor-induced puerarin accumulation in transgenic cells, which indicated that SA inhibited JA biosynthesis in the cells and that SA might be used as a substitute for JA to mediate the elicitor- and NO-induced puerarin biosynthesis. It was, therefore, concluded that NO might mediate the elicitor-induced puerarin biosynthesis through SA- and JA-dependent signal pathways in wildtype P. thomsonii Benth. cells and transgenic NahG cells respectively.

Cell Line↗
PubMed2001

Biosynthesis of menaquinone (vitamin K2) and ubiquinone (coenzyme Q): a perspective on enzymatic mechanisms.

The benzoquinone ubiquinone (coenzyme Q) and the naphthoquinones menaquinone (vitamin K2) and demethylmenaquinone are derived from the shikimate pathway, which has been described as a "metabolic tree with many branches." Menaquinone (MK) is considered a vitamin, but coenzyme (Q) is not; MK is an essential nutrient (it cannot be synthesized by mammals), whereas Q is not considered an essential nutrient since it can be synthesized from the amino acid tyrosine. The quinone nucleus of Q is derived directly from chorismate, whereas that of MK is derived from chorismate via isochorismate. The prenyl side chain of both quinones is derived from prenyl diphosphate, and the methyl groups are derived from S-adenosylmethionine. MK biosynthesis requires 2-ketoglutarate and the cofactors ATP, coenzyme A (CoASH), and thiamine pyrophosphate. In spite of the fact that both quinones originate from the shikimate pathway, there are important differences in their biosynthesis. In MK biosynthesis, the prenyl side chain is introduced in the next to last step, which is accompanied by loss of the carboxyl group, whereas in Q biosynthesis, the prenyl side chain is introduced at the second step, with retention of the carboxyl group. In MK biosynthesis, all the reactions of the pathway up to the prenylation (next to last step) are carried out by soluble enzymes, whereas all the enzymes involved in Q biosynthesis except the first are membrane bound. In MK biosynthesis the last step is a C-methylation; in Q biosynthesis, the last step is an O-methylation. In Q biosynthesis a second C-methylation and O-methylation take place in the middle part of the pathway. In spite of the fact that Q and MK biosynthesis diverges at chorismate, the C-methylations involved in both pathways are carried out by the same enzyme. Finally, Q biosynthesis under aerobic conditions requires molecular oxygen; anaerobic biosynthesis of Q and MK incorporates oxygen atoms derived from water. The current status of the pathways with particular emphasis on the reaction mechanisms, is discussed in this review.

Escherichia coli↗
PubMed2001

The potential mechanism for glutamine-induced collagen biosynthesis in cultured human skin fibroblasts.

Although glutamine (Gln) is known as an important stimulator of collagen biosynthesis in collagen-producing cells, the mechanism and endpoints by which it regulate the process remain largely unknown. Intermediates of Gln interconversion: glutamate (Glu) and pyrroline-5-carboxylate (P5C) stimulate collagen biosynthesis in cultured cells but evoke different maxima of collagen biosynthesis stimulating activity at different times of incubation. P5C was found to be the most potent stimulator of collagen biosynthesis after 6 h of incubation (approx. three-fold increase); after 12 h, it induced increase in collagen biosynthesis to 260%, while at 24 h, the process was decreased to approximately 80% of control values. Glu induced increase in collagen biosynthesis to approximately 180%, 400% and 120% of control values, after 6, 12 and 24 h, respectively, suggesting that after 12 h of incubation, Glu was the most potent stimulator of collagen biosynthesis. Glu was also the most potent stimulator of type I procollagen expression at this time. After 6, 12 and 24 h incubation, Gln induced collagen biosynthesis to approximately 112, 115 and 230% of control values, respectively. Since prolidase is known to be involved in collagen metabolism, the enzyme activity assay was performed in fibroblasts cultured in the presence of Gln, Glu and P5C. While Gln and Glu required 24 h for maximal stimulation of prolidase activity, P5C induced it after 6-12 h. The data suggest that P5C induced collagen biosynthesis and prolidase activity in a shorter time than Gln and Glu. We considered that P5C directly stimulates the processes, while Gln acts through its intermediate-P5C. Reduction of P5C to proline is coupled to the conversion of glucose-6-phosphate (G6P) to 6-phospho-gluconate, catalyzed by G6P dehydrogenase. We have found that dehydroepiandrosterone (DHEA), a potent inhibitor of G6P dehydrogenase, inhibited a stimulatory effect of P5C on collagen synthesis, expression of type I collagen and prolidase activity. Our results postulate a potential mechanism of glutamine-induced collagen biosynthesis through its intermediate - P5C. P5C-dependent activation of nucleotide biosynthesis, prolidase activity and P5C conversion into proline may contribute to the stimulation of collagen biosynthesis.

Cells, Cultured↗
PubMed1998

A distinct difference in the metabolic stimulus-response coupling pathways for regulating proinsulin biosynthesis and insulin secretion that lies at the level of a requirement for fatty acyl moieties.

The regulation of proinsulin biosynthesis in pancreatic beta-cells is vital for maintaining optimal insulin stores for glucose-induced insulin release. The majority of nutrient fuels that induce insulin release also stimulate proinsulin biosynthesis, but since insulin exocytosis and proinsulin synthesis involve different cellular mechanisms, a point of divergence in the respective metabolic stimulus-response coupling pathways must exist. A parallel examination of the metabolic regulation of proinsulin biosynthesis and insulin secretion was undertaken in the same beta-cells. In MIN6 cells, glucose-induced proinsulin biosynthesis and insulin release shared a requirement for glycolysis to generate stimulus-coupling signals. Pyruvate stimulated both proinsulin synthesis (threshold 0.13-0.2 mM) and insulin release (threshold 0.2-0.3 mM) in MIN6 cells, which was eliminated by an inhibitor of pyruvate transport (1 mM alpha-cyano-4-hydroxycinnamate). A combination of alpha-oxoisohexanoate and glutamine also stimulated proinsulin biosynthesis and insulin release in MIN6 cells, which, together with the effect of pyruvate, indicated that anaplerosis was necessary for instigating secondary metabolic stimulus-coupling signals in the beta-cell. A consequence of increased anaplerosis in beta-cells is a marked increase in malonyl-CoA, which in turn inhibits beta-oxidation and elevates cytosolic fatty acyl-CoA levels. In the beta-cell, long-chain fatty acyl moieties have been strongly implicated as metabolic stimulus-coupling signals for regulating insulin exocytosis. Indeed, it was found in MIN6 cells and isolated rat pancreatic islets that exogenous oleate, palmitate and 2-bromopalmitate all markedly potentiated glucose-induced insulin release. However, in the very same beta-cells, these fatty acids in contrast inhibited glucose-induced proinsulin biosynthesis. This implies that neither fatty acyl moieties nor beta-oxidation are required for the metabolic stimulus-response coupling pathway specific for proinsulin biosynthesis, and represent an early point of divergence of the two signalling pathways for metabolic regulation of proinsulin biosynthesis and insulin release. Therefore alternative metabolic stimulus-coupling factors for the specific control of proinsulin biosynthesis at the translational level were considered. One possibility examined was an increase in glycerophosphate shuttle activity and change in cytosolic redox state of the beta-cell, as reflected by changes in the ratio of alpha-glycerophosphate to dihydroxyacetone phosphate. Although 16.7 mM glucose produced a significant rise in the alpha-glycerophosphate/dihydroxyacetone phosphate ratio, 1 mM pyruvate did not. It follows that the cytosolic redox state and fatty acyl moieties are not necessarily involved as secondary metabolic stimulus-coupling factors for regulation of proinsulin biosynthesis. However, the results indicate that glycolysis and the subsequent increase in anaplerosis are indeed necessary for this signalling pathway, and therefore an extramitochondrial product of beta-cell pyruvate metabolism (that is upstream of the increased cytosolic fatty acyl-CoA) acts as a key intracellular secondary signal for specific control of proinsulin biosynthesis by glucose at the level of translation.

Animals↗
PubMed1997

In vivo and in vitro effects of thiolactomycin on fatty acid biosynthesis in Streptomyces collinus.

A stable-isotope assay was used to analyze the effectiveness of various perdeuterated short-chain acyl coenzyme A (acyl-CoA) compounds as starter units for straight- and branched-chain fatty acid biosynthesis in cell extracts of Streptomyces collinus. In these extracts perdeuterated isobutyryl-CoA was converted to isopalmitate (a branched-chain fatty acid), while butyryl-CoA was converted to palmitate (a straight-chain fatty acid). These observations are consistent with previous in vivo analyses of fatty acid biosynthesis in S. collinus, which suggested that butyryl-CoA and isobutyryl-CoA function as starter units for palmitate and isopalmitate biosynthesis, respectively. Additionally, in vitro analysis demonstrated that acetyl-CoA can function as a starter unit for palmitate biosynthesis. Palmitate biosynthesis and isopalmitate biosynthesis in these cell extracts were both effectively inhibited by thiolactomycin, a known type II fatty acid synthase inhibitor. In vivo experiments demonstrated that concentrations of thiolactomycin ranging from 0.1 to 0.2 mg/ml produced both a dramatic decrease in the cellular levels of branched-chain fatty acids and a surprising three- to fivefold increase in the cellular levels of the straight-chain fatty acids palmitate and myristate. Additional in vivo incorporation studies with perdeuterated butyrate suggested that, in accord with the in vitro studies, the biosynthesis of the palmitate from butyryl-CoA decreases in the presence of thiolactomycin. In contrast, in vivo incorporation studies with perdeuterated acetate demonstrated that the biosynthesis of palmitate from acetyl-CoA increases in the presence of thiolactomycin. These observations clearly demonstrate that isobutyryl-CoA is a starter unit for isopalmitate biosynthesis and that either acetyl-CoA or butyryl-CoA can be a starter unit for palmitate biosynthesis in S. collinus. However, the pathway for palmitate biosynthesis from acetyl-CoA is less sensitive to thiolactomycin, and it is suggested that the basis for this difference is in the initiation step.

Acetic Acid↗
PubMed1983

Endogenous biosynthesis of prostacyclin and thromboxane and platelet function during chronic administration of aspirin in man.

To assess the pharmacologic effects of aspirin on endogenous prostacyclin and thromboxane biosynthesis, 2,3-dinor-6-keto PGF1 alpha (PGI-M) and 2,3-dinor-thromboxane B2 (Tx-M) were measured in urine by mass spectrometry during continuing administration of aspirin. To define the relationship of aspirin intake to endogenous prostacyclin biosynthesis, sequential urines were initially collected in individuals prior to, during, and subsequent to administration of aspirin. Despite inter- and intra-individual variations, PGI-M excretion was significantly reduced by aspirin. However, full mass spectral identification confirmed continuing prostacyclin biosynthesis during aspirin therapy. Recovery of prostacyclin biosynthesis was incomplete 5 d after drug administration was discontinued. To relate aspirin intake to indices of thromboxane biosynthesis and platelet function, volunteers received 20 mg aspirin daily followed by 2,600 mg aspirin daily, each dose for 7 d in sequential weeks. Increasing aspirin dosage inhibited Tx-M excretion from 70 to 98% of pretreatment control values; platelet TxB2 formation from 4.9 to 0.5% and further inhibited platelet function. An extended study was performed to relate aspirin intake to both thromboxane and prostacyclin generation over a wide range of doses. Aspirin, in the range of 20 to 325 mg/d, resulted in a dose-dependent decline in both Tx-M and PGI-M excretion. At doses of 325-2,600 mg/d Tx-M excretion ranged from 5 to 3% of control values while PGI-M remained at 37-23% of control. 3 d after the last dose of aspirin (2,600 mg/d) mean Tx-M excretion had returned to 85% of control, whereas mean PGI-M remained at 40% of predosing values. Although the platelet aggregation response (Tmax) to ADP ex vivo was inhibited during administration of the lower doses of aspirin the aggregation response returned to control values during the final two weeks of aspirin administration (1,300 and 2,600 mg aspirin/d) despite continued inhibition of thromboxane biosynthesis. These results suggest that although chronic administration of aspirin results in inhibition of endogenous thromboxane and prostacyclin biosynthesis over a wide dose range, inhibition of thromboxane biosynthesis is more selective at 20 than at 2,600 mg aspirin/d. However, despite this, inhibition of platelet function is not maximal at the lower aspirin dosage. Doses of aspirin in excess of 80 mg/d resulted in substantial inhibition of endogenous prostacyclin biosynthesis. Thus, it is unlikely that any dose of aspirin can maximally inhibit thromboxane generation without also reducing endogenous prostacyclin biosynthesis. These results also indicate that recovery of endogenous prostacyclin biosynthesis is delayed following aspirin administration and that the usual effects of aspirin on platelet function ex vivo may be obscured during chronic aspirin administration in man.

Adenosine Diphosphate↗
PubMed1996

Glucose-regulated translational control of proinsulin biosynthesis with that of the proinsulin endopeptidases PC2 and PC3 in the insulin-producing MIN6 cell line.

In the short term (< 2 h), proinsulin biosynthesis is predominately glucose regulated at the translational level; however, the details at the molecular level behind this mechanism are not well defined. One of the major hindrances for gaining a better understanding of the proinsulin biosynthetic mechanism has been a lack of an abundant source of beta-cells that express a phenotype of regulated proinsulin biosynthesis in the appropriate 2.8-16.7 mmol/l glucose range as defined in normal pancreatic islets. In this study, we demonstrate that in the MIN6 cell line, specific glucose-regulated translational control of proinsulin biosynthesis is present in the appropriate glucose concentration range. In addition to that of proinsulin, the biosynthesis of the two proinsulin conversion endopeptidases, PC2 and PC3, was coordinately glucose regulated in MIN6 cells, whereas that of the exopeptidase, carboxypeptidase H, was unaffected by glucose. Proinsulin, PC2 and PC3 biosynthesis was specifically stimulated over that of total MIN6 cell protein synthesis above a threshold of 4 mmol/l glucose that reached a maximum rate between 8 and 10 mmol/l glucose. Glucose-induced proinsulin, PC2, and PC3 biosynthesis was rapid (occurring after a 20-min lag period but reaching a maximum by 60 min), unaffected by the presence of actinomycin D; and in parallel experiments, stimulatory glucose concentrations did not alter MIN6 cell total preproinsulin, PC2, or PC3 mRNA levels. Thus, short-term (< 2 h) glucose stimulation of proinsulin, PC2 and PC3 biosynthesis in MIN6 cells, like that in isolated islets, was mediated at the translational level. Intracellular signals for mediating glucose-stimulated proinsulin PC2 and PC3 biosynthesis translation in MIN6 cells also appeared to be similar to those in pancreatic islets, requiring glucose metabolism and a supporting role for protein kinase A. However, protein kinase C or a Ca(2+)-dependent protein kinase did not appear to be required for glucose-regulated proinsulin biosynthesis in MIN6 cells, as in islets. MIN6 cells are the first beta-cell line that indicate glucose-regulated proinsulin biosynthesis translation essentially identical to that in differentiated islet beta-cells and will be an important experimental model to better define the mechanism of proinsulin biosynthesis in detail.

Animals↗
PubMed1978

The effect of sugars on (pro)insulin biosynthesis.

Rates of incorporation of [4,5-(3)H]leucine into insulin plus proinsulin, designated ;(pro)insulin', and total protein in rat pancreatic islets were measured. Glucose stimulates rates of total protein and (pro)insulin biosynthesis, but (pro)insulin biosynthesis is stimulated preferentially. Mannose and N-acetylglucosamine also stimulate (pro)insulin and total protein biosynthesis; inosine and dihydroxyacetone stimulate (pro)insulin biosynthesis specifically. Fructose does not stimulate (pro)insulin biosynthesis when tested alone, but does so in the presence of low concentrations of glucose, mannose or N-acetylglucosamine. Many glucose analogues do not stimulate (pro)insulin biosynthesis. Mannoheptulose inhibits synthesis of (pro)insulin and total protein stimulated by glucose or mannose but not by dihydroxyacetone, inosine or N-acetylglucosamine; phloretin (9mum) inhibits N-acetylglucosamine-stimulated (pro)insulin biosynthesis preferentially. The data are in agreement with the view that the same glucose-sensor mechanism may control both insulin release and biosynthesis, and ;substrate-site' model is suggested. The threshold for stimulation of biosynthesis of (pro)insulin and total protein is lower than that found for glucose-stimulated insulin release; moreover the biosynthetic response to an elevation of glucose concentration is slower than that found for insulin release. The physiological implication of these findings is discussed. Caffeine and isobutylmethylxanthine, at concentrations known to increase islet 3':5'-cyclic AMP and potentiate glucose-induced insulin release, were without effect on rates of glucose-stimulated (pro)insulin biosynthesis.

Adenosine↗
PubMed1996

Regulation of cardiolipin biosynthesis in the heart.

Cardiolipin is one of the principle phospholipids in the mammalian heart comprising as much as 15-20% of the entire phospholipid phosphorus mass of that organ. Cardiolipin is localized primarily in the mitochondria and appears to be essential for the function of several enzymes of oxidative phosphorylation. Thus, cardiolipin is essential for production of energy for the heart to beat. Cardiac cardiolipin is synthesized via the cytidine-5'-diphosphate-1,2-diacyl-sn-glycerol pathway. The properties of the four enzymes of the cytidine-5'-diphosphate-1,2-diacyl-sn-glycerol pathway have been characterized in the heart. The rate-limiting step of this pathway is catalyzed by the phosphatidic acid: cytidine-5'-triphosphate cytidylyltransferase. Several regulatory mechanisms that govern cardiolipin biosynthesis in the heart have been uncovered. Current evidence suggests that cardiolipin biosynthesis is regulated by the energy status (adenosine-5'-triphosphate and cytidine-5'-triphosphate level) of the heart. Thyroid hormone and unsaturated fatty acids may regulate cardiolipin biosynthesis at the level of three key enzymes of the cytidine-5'-diphosphate-1,2-diacyl-sn-glycerol pathway, phosphatidylglycerol phosphate synthase, phosphatidyl-glycerolphosphate phosphatase and cardiolipin synthase. Newly synthesized phosphatidic acid and phosphatidylglycerol may be preferentially utilized for cardiolipin biosynthesis in the heart. In addition, separate pools of phosphatidylglycerol, including an exogenous (extra-mitochondrial) pool not derived from de novo phosphatidylglycerol biosynthesis, may be utilized for cardiac cardiolipin biosynthesis. In several mammalian tissues a significant number of studies on polyglycerophospholipid biosynthesis have been documented, including detailed studies in the lung and liver. However, in spite of the important role of cardiolipin in the maintenance of mitochondrial function and membrane integrity, studies on the control of cardiolipin biosynthesis in the mammalian heart have been largely neglected. The purpose of this review will be to briefly discuss cardiolipin and cardiolipin biosynthesis in some selected model systems and focus primarily on current studies involving the regulation of cardiolipin biosynthesis in the heart.

Animals↗
PubMed1999

Juvenile hormone biosynthesis in larval and adult stick insects, Carausius morosus.

Corpora allata (CA) from adult egg-carrying Indian stick insects, Carausius morosus, synthesise and release juvenile hormone (JH) III in vitro. No JH biosynthesis was observed in larvae, young adults, and old adult females that do not carry sclerotised eggs. In females, which bear sclerotised eggs, a consistent JH biosynthesis was observed. Supplementation of precursors of JH biosynthesis (farnesol, mevalonic acid lactone) greatly enhanced JH biosynthesis in a stage-, age-, and dose-dependent manner, but CA from the last larval instar retained the biosynthesised JH within the gland. Elevated calcium concentration in the incubation medium stimulated JH biosynthesis by CA from older adults but had either no or a poor effect on CA from young adults and larvae. The results obtained with farnesol, mevalonic acid lactone, and calcium indicate that the rate-limiting steps of JH biosynthesis very likely occur before the formation of mevalonic acid and that these early steps cannot be stimulated by elevated calcium concentrations in larvae and young adults. In older adults, in which spontaneous JH biosynthesis occurs, elevated calcium concentration can markedly stimulate JH biosynthesis. A pre-purified extract from brains of adult females had a stimulating effect on JH biosynthesis by CA from adult females. The results indicate that JH biosynthesis in C. morosus may require food-derived farnesol and may be regulated by allatotropic signals from the brain, possibly triggered by sclerotised oocytes in the ovary.

Journal Article↗
PubMed1982

Prostaglandin biosynthesis in the human fetal adrenal gland: regulation by glucocorticosteroids.

Human fetal adrenal (HFA) tissue was maintained in organ culture to evaluate the biosynthesis of prostaglandins and hormonal regulation of prostaglandin formation by this tissue. The HFA tissue secreted substantial amounts of prostaglandin E(2), prostaglandin F(2alpha), 13,14-dihydro-15-ketoprostaglandin F(2alpha), 6-ketoprostaglandin F(1alpha), and thromboxane B(2); secretion of prostaglandin D(2) could not be demonstrated. Prostaglandin biosynthesis in HFA tissue was inhibited in a time-dependent manner by corticotropin (ACTH; 0.4 muM); by the fourth day of culture, the extent of inhibition of biosynthesis of each prostaglandin was 60-90%. Progesterone (1 muM), cortisol (1 muM), and dexamethasone (1 muM) inhibited prostaglandin biosynthesis whereas estradiol (1 muM) did not. Of the compounds tested for inhibitory activity, dexamethasone was the most potent. An inhibitor of 11beta-hydroxylase activity (metyrapone; 0.1 mM) effectively eliminated the inhibition of prostaglandin biosynthesis caused by corticotropin and progesterone. Metyrapone treatment alone caused a 3-fold increase in prostaglandin biosynthesis by fetal adrenal tissues. Similar stimulatory effects resulted from treatment with inhibitors of (i) 3beta-hydroxysteroid dehydrogenase (cyanoketone; 15 muM), (ii) steroid 17alpha-hydroxylase (SU 10603; 19 muM), and (iii) cholesterol side-chain cleavage (aminoglutethimide; 1 mM). Inhibition of prostaglandin biosynthesis by dexamethasone in the presence or absence of metyrapone was concentration dependent and 50% inhibition could be demonstrated at 1 nM. A competitive inhibitor of the binding of glucocorticosteroids to cytoplasmic receptors (cortisol 21-mesylate; 1 muM) significantly reduced the inhibition of prostaglandin biosynthesis effected by dexamethasone (10 nM). These findings suggest that prostaglandin biosynthesis in the HFA gland is regulated by endogenously synthesized glucocorticosteroids, the actions of which are mediated by a glucocorticosteroid receptor. Such glucocorticosteroids induce the synthesis of a substance that inhibits prostaglandin biosynthesis.

Adrenal Glands↗
PubMed1997

Sauvagine and TRH differentially stimulate proopiomelanocortin biosynthesis in the Xenopus laevis intermediate pituitary.

In the amphibian Xenopus laevis, adaptation of the skin color to background light intensity is regulated by alpha-melanophore-stimulating hormone (alpha-MSH), a proopiomelanocortin (POMC)-derived peptide. In animals adapted to a white background, the level of POMC biosynthesis in the intermediate pituitary is much lower than in animals adapted to a black background. Release of alpha-MSH from neurointermediate lobes of white-adapted animals is stimulated in vitro by the regulatory peptides sauvagine and thyrotropin-releasing hormone (TRH), which are produced in the magnocellular nucleus of the hypothalamus. To study the role of sauvagine, cAMP, TRH and phorbol 12-myristate 13-acetate (PMA) in the regulation of POMC biosynthesis, the degree of incorporation of radioactive amino acids into the POMC protein was determined after treatment of the neurointermediate lobes with these secretagogues. When lobes of white-adapted animals are incubated in vitro, biosynthetic activity spontaneously increases because hypothalamic inhibitory control is removed by dissection. In addition to this control situation, the effects of secretagogues were tested on lobes with an inhibited level of biosynthesis, which is achieved by addition of neuropeptide Y (NPY) to the incubation medium. After 24 h of treatment, TRH stimulated POMC biosynthesis in NPY-inhibited lobes of white-adapted animals from 40.2 to 95.3% of control level. This stimulation could not be reduced by adding PMA, which indicates that protein kinase C is not involved in the stimulation of POMC biosynthesis by TRH. Sauvagine partially restored POMC biosynthesis from 27.2 to 62.5% of control level, whereas 8-Br-cAMP completely counteracted NPY inhibition from 27.8 to 97.5% of control level. After 3 days of treatment, stimulation by sauvagine and 8-Br-cAMP was maintained (sauvagine increased POMC biosynthesis in NPY-inhibited lobes from 7.4 to 36.2% of control level and 8-Br-cAMP stimulated from 6.5 to 82.5% of control level). TRH had no effect on POMC biosynthesis after 3 days of treatment, although its receptor was still functional as was shown in superfusion experiments where TRH stimulated alpha-MSH secretion. The observations indicate that the neuropeptides sauvagine and TRH differently control POMC biosynthesis in the Xenopus intermediate pituitary. This differential regulation is not only apparent with regard to time aspects (sauvagine has a sustained regulatory function, whereas TRH is only effective in the initial phase of POMC biosynthesis stimulation), but also an uncoupling of biosynthetic and release processes could be shown for TRH, which did not occur with sauvagine.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1987

Interleukin 1 dose-dependently affects the biosynthesis of (pro)insulin in isolated rat islets of Langerhans.

Human crude and recombinant interleukin 1 (IL-1) was found to dose- and time-dependently affect the biosynthesis of (pro)insulin in isolated rat islets of Langerhans. Incubation of rat islets with either 0.5 U/ml or 5 U/ml of crude IL-1 for 1 h had no detectable effect on (pro)insulin biosynthesis. After 24 hours of exposure 0.5 U/ml of crude or 0.6 ng/ml of recombinant IL-1 (beta) increased the (pro)insulin biosynthesis by 42% and 58%, respectively, whereas a 10-fold greater concentration of IL-1 decreased the (pro)insulin biosynthesis by 74% and 89%, respectively. The increase in (pro)insulin biosynthesis was accompanied by an increase in total protein biosynthesis indicating a nonspecific stimulatory action of low IL-1 concentrations. In contrast, high IL-1 concentrations caused a more selective decrease of the (pro) insulin biosynthesis when compared to the total protein biosynthesis. In addition, low IL-1 concentrations were found to increase and high concentrations to decrease the relative levels of pre-proinsulin mRNA suggesting that IL-1 may act both at a pre- and post-translational level of insulin biosynthesis.

Animals↗
PubMed2002

Succinate is a preferential metabolic stimulus-coupling signal for glucose-induced proinsulin biosynthesis translation.

The secondary signals emanating from increased glucose metabolism, which lead to specific increases in proinsulin biosynthesis translation, remain elusive. It is known that signals for glucose-stimulated insulin secretion and proinsulin biosynthesis diverge downstream of glycolysis. Consequently, the mitochondrial products ATP, Krebs cycle intermediates, glutamate, and acetoacetate were investigated as candidate stimulus-coupling signals specific for glucose-induced proinsulin biosynthesis in rat islets. Decreasing ATP levels by oxidative phosphorylation inhibitors showed comparable effects on proinsulin biosynthesis and total protein synthesis. Although it is a cofactor, ATP is unlikely to be a metabolic stimulus-coupling signal specific for glucose-induced proinsulin biosynthesis. Neither glutamic acid methyl ester nor acetoacetic acid methyl ester showed a specific effect on glucose-stimulated proinsulin biosynthesis. Interestingly, among Krebs cycle intermediates, only succinic acid monomethyl ester specifically stimulated proinsulin biosynthesis. Malonic acid methyl ester, an inhibitor of succinate dehydrogenase, also specifically increased glucose-induced proinsulin biosynthesis without affecting islet ATP levels or insulin secretion. Glucose caused a 40% increase in islet intracellular succinate levels, but malonic acid methyl ester showed no further effect, probably due to efficient conversion of succinate to succinyl-CoA. In this regard, a GTP-dependent succinyl-CoA synthetase activity was found in cytosolic fractions of pancreatic islets. Thus, succinate and/or succinyl-CoA appear to be preferential metabolic stimulus-coupling factors for glucose-induced proinsulin biosynthesis translation.

Adenosine Triphosphate↗
PubMed1994

Mechanism by which ethanol inhibits phosphatidylcholine biosynthesis in human leukemic monocyte-like U937 cells.

A previous study showing that ethanol (ETOH) blocked [3H]choline incorporation into phosphatidylcholine (PC) suggested an inhibition of PC biosynthesis in human leukemic monocyte-like U937 cells. The mechanism of the inhibitory action of ETOH was investigated. Cells were pulsed with [3H]choline for 30 min and chased in the presence or absence of ETOH for up to 6 h. PC biosynthesis was inhibited drastically within 1 h after exposure to ETOH which increased intracellular cAMP appreciably. After a 3-h treatment, ETOH significantly inhibited both choline kinase (CK) and the cytosolic CTP: cholinephosphate cytidylyltransferase (CT). The inactivated CT was no longer stimulated by exogenous phosphatidylglycerol (PG). There was no evidence for redistribution of CT activity between cytosol and microsomes. When cells were exposed to 8-Bromo-cAMP ranging from 100 to 300 microM, PC biosynthesis remained unaffected despite the drastically elevated cAMP. These results seem to suggest that the raised cAMP is not a prerequisite for the inhibition of PC biosynthesis in U937 cells. Following pretreatment with protein kinase inhibitors (H-89 and K-252a), PC biosynthesis was decreased significantly and the inhibitory effect of ETOH was potentiated. Taken together, our results suggest that the inhibition of PC biosynthesis and the inhibitory effect of ETOH are independent of the activation of cAMP-dependent protein kinase. Unlike protein kinase inhibitors, pretreatment with tyrosine kinase inhibitors (erbstatin, genistein and tyrphostin 25) resulted in differential effects on PC biosynthesis and on the inhibitory action of ETOH. Genistein stimulated PC biosynthesis by 30 per cent as well as partially preventing/reversing the ETOH action, while tyrphostin 25 produced a synergistic inhibition. The relevance of tyrosine phosphorylation/dephosphorylation to the regulation of PC biosynthesis and ETOH action remains to be established.

8-Bromo Cyclic Adenosine Monophosphate↗