Brefeldin A: a specific inhibitor of cell wall polysaccharide biosynthesis in oat coleoptile segments

The effect of brefeldin A (BFA) on the synthesis and incorporation of polysaccharides, proteins and glycoproteins into the cell wall of subapical coleoptile segments isolated from etiolated oat seedlings (Avena sativa L. cv. Angelica) has been investigated. In the presence of D-[U-¹⁴C]-glucose, the incorporation of radioactive glycosyl residues into buffer-soluble, membrane (matrix polysaccharides) and cell wall polysaccharides was drastically inhibited by increasing concentrations of BFA up to 10 μ·mL⁻¹. BFA also altered the pattern of these polysaccharides suggesting a different sensitivity of glycosyltransferases toward the action of the drug. The incorporation of [U-¹⁴C]-glycine or L-[U-¹⁴C]-leucine into non-covalently- and covalently-bound cell wall proteins as well as the incorporation of radioactive N-acetylglucosamine residues into the newly synthesised oligosaccharidic chains of cytosolic, membrane and cell wall glycoproteins remained unchanged in the presence of 10 μg·mL⁻¹ BFA. The data demonstrate that, in oat coleoptile segments, BFA specifically inhibits the synthesis of cellulose and matrix polysaccharides without altering the synthesis and incorporation of proteins and glycoproteins into the cell wall. In addition, it is demonstrated that BFA does not affect the in vivo activity of glycosyltransferases involved in the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to the oligosaccharidic chains of glycoproteins.     

Utilization of exogenously supplied primary precursors for essential oil synthesis in cymbopogon species

The relative efficiency of incorporation of the exogenously supplied primary precursors [2-¹⁴C]acetate, [U-¹⁴C]glucose and [U-¹⁴C]sucrose into essential oil was determined in the immature leaves of threeCymbopogon species(C. martinii,C. winterianus andC. flexuosus). Acetate was most efficiently incorporated into essential oil inC. winterianus andC. flexuosus, whereas glucose was the best precursor inC. martinii. The observations are consistent when expressed as radioactivity [Bq] of essential oil per leaf, as percent incorporation or moles of precursors utilized for essential oil synthesis. Thus, there is selectivity in the efficiency of precursor utilization for the oil synthesis inCymbopogon species.     

Further investigations of the incorporation of [1-14C]acetate into the lipids of the silkworm Bombyx mori L

1. After the injection of sodium [1-¹⁴C]acetate, the highest incorporation of ¹⁴C into the lipids of the silkworm was observed after 24hr. 2. The specific radioactivity of the palmitic acid fraction was greater and increased more rapidly than that of the stearic acid fraction, which was consistent with the precursor–product relationship to be expected on the basis of current concepts of fatty acid synthesis in vivo. 3. The results indicate the probability of synthesis of lipid components in tissues other than the fat body. 4. Fractionation studies indicate considerable differences in the rate of incorporation of [1-¹⁴C]acetate into neutral lipids and phospholipids between larvae and pupae as well as among tissues of larvae. 5. The rate of incorporation of [1-¹⁴C]acetate remains constant throughout pupal development.     

In vivo and in vitro inhibition of lipid-linked saccharide and glycoprotein synthesis in plants by amphomycin

The effect of the polypeptide antibiotic, amphomycin, on the in vitro and in vivo synthesis of polyprenyl-linked sugars and glycoproteins in plants was examined. This antibiotic blocked the transfer of mannose from GDP-[¹⁴C]mannose into mannosyl-phos-phoryl-dolichol by a particulate enzyme preparation from mung beans and also inhibited the transfer of GlcNAc from UDP-[³H]GlcNAc to GlcNAc-pyrophosphoryl-polyisoprenol. The in vitro incorporation of these sugars into trichloroacetic acid-insoluble material was also markedly inhibited by this antibiotic. Since most of the radioactivity incorporated into this insoluble material is rendered water-soluble by treatment with pronase, it seems likely that these sugars are incorporated into glycoproteins whose synthesis is sensitive to amphomycin. Amphomycin also inhibited the transfer of glucose from UDP-[¹⁴C]glucose to steryl glucosides, although this system was less sensitive to antibiotic than was synthesis of the polyprenyl-linked sugars. The antibiotic did not block the in vitro transfer of glucose from UDP-[¹⁴C]glucose to β-glucans. In carrot slice cultures, amphomycin also inhibited the incorporation of [¹⁴C]mannose into glycolipid and glycoprotein, but it did not prevent the incorporation of [¹⁴C]lysine into protein.     

Alterations of mitochondrial protein metabolism in liver, brown adipose tissue and skeletal muscle during cold-acclimation

Incorporation of -[U-¹⁴C] leucine into liver, brown adipose tissue and skeletal muscle mitochondrial proteins was determined in vivo and in vitro during cold-acclimation. Major alterations in mitochondrail protein metabolism were observed in brown adipose tissue and skeletal muscle but not in liver. Immediate cold-exposure is accompanied by an inhibition of the in vivo incorporation of -[U-¹⁴C] leucine into mitochondrial proteins of all tissues. However, during cold-acclimation the incorporation of leucine increases markedly in brown adipose tissue, continues to decrease in skeletal muscle, nut does not change appreciably in the liver. Because increased incorporation of -[U-¹⁴C] leucine into brown adipose tissue mitochondrial proteins was observed both in vivo and in vitrom it can be concluded that the mitochondrial protein-synthesizing system of this tissue is directly affected by the acclimation process. The observed changes in mitochondrial protein metabolism of brown adipose tissue and skeletal muscle might be responsible for the development of several morphological and biochemical alterations that characterize the establishment in these tissues of the cold-acclimated state.     

Effects of Filipin and cholesterol on housefly, Musca domestica L., and wax moth, Galleria mellonella L

The effects of filipin on insects are dependent on the molar ratio of cholesterol to filipin. The larvicidal effects of the polyene antibiotic, filipin, can be prevented by excess cholesterol (“excess” herein is defined as a molar ratio of cholesterol to filipin of greater than 2 : 1) in housefly, Musca domestica L., and wax moth, Galleria mellonella L., larvae. Excess cholesterol also prevents the chemosterilant effect of filipin in housefly adults. The filipin-induced inhibition of [¹⁴C]cholesterol uptake by wax moth larvae is prevented by excess cholesterol; cholesterol uptake is increased severalfold. Dietary filipin, in the absence of added cholesterol, caused loss of ³²P from housefly tissues and decreased the incorporation of ³²P- and [¹⁴C]methyl-labeled choline into phospholipids of wax moth tissues. Addition of excess cholesterol to filipin-containing diets enhanced incorporation of ³²P into the different classes of phospholipids, and phospholipid synthesis was nearly doubled.     

Protein and nucleic acid synthesis during imbibition of dormant and after-ripened Vaccaria pyramidata seeds.

The regulation of nucleic acid and protein synthesis in dormant, thermodormant, and after-ripened embryos of Vaccaria pyramidata (Caryophyllaceae) has been studied. Germination of after-ripened V. pyramidata seeds is prevented by inhibitors of protein, RNA, and DNA synthesis. The synthesis of both protein and RNA is activated at the beginning of imbibition, whereas [³H]thymidine incorporation does not start until the second period of the imbibition phase. [³H]Thymidine incorporation is greatly reduced in embryos treated with cycloheximide or 6-methylpurine. There is no correlation between the level of [³H]uracil and l-[¹⁴C]leucine incorporation into macromolecules and the physiological state of the seeds: tRNA, ribosomal RNA, and poly(A)-containing RNA (probably mRNA) as well as proteins are synthesized at the same rate in both dormant and thermodormant embryos as in after-ripened embryos. The protein patterns of dormant and after-ripened embryos are similar, as shown by electrophoresis and electrofocusing of double-labeled proteins. The level of DNA synthesis, measured as [³H]thymidine incorporation, may, on the other hand, indicate the physiological activity of the seeds: [³H]Thymidine is incorporated at a high rate in after-ripened embryos only and remains at a low level in dormant or thermodormant embryos. This correlation is, however, observed only in the axes. DNA synthesis in the cotyledons does not show any relation to the developmental stage of the seeds. These results are discussed in relation to the regulation of dormancy and after-ripening of seeds.     

The use of exogenous δ-aminolaevulinic acid for the studies of the regulation of haem synthesis in rabbit reticulocytes

δ-Amino [4-¹⁴C]laevulinate added to reticulocytes incubated in vitro is incorporated into haem. Exogenous δ-aminolaevulinate restores the incorporation of ⁵⁹Fe into haem in reticulocytes which had been treated with isonicotinic acid hydrazide (INH) or penicillamine and were hence unable to synthesize δ-aminolaevulinate. On the other hand, the addition of δ-aminolaevulinate does not restore the incorporation of Fe into reticulocytes incubated with haemin. The inhibition of the incorporation of iron is neither restored by δ-aminolaevulinate in reticulocytes incubated with cycloheximide (which inhibits globin synthesis and thus elevates the free intracellular haem pool). These results suggest that in intact reticulocytes haemin does not inhibit δ-aminolaevulinate synthetase. This conclusion is further supported by the finding that the pattern of incorporation of [2-¹⁴C]glycine and δ-amino[4-¹⁴C]-laevulinate into haem differs in reticulocytes incubated with an inhibitor of δ-aminolaevulinate synthetase (INH) and in reticulocytes incubated with haemin and cycloheximide.     

Effect of pyridoxine deficiency on nucleic acid metabolism in the rat

1. The nucleic acid metabolism in the pyridoxine-deficient rat has been investigated through studies on the incorporation of radioactivity from various isotopically labelled compounds into liver and spleen DNA and RNA. 2. In pyridoxine deficiency, the incorporation of radioactivity from sodium [¹⁴C]formate was apparently increased. The magnitude of this effect on incorporation into liver RNA and DNA and spleen RNA was approximately the same. The incorporation into spleen DNA was enhanced to a much greater degree. Administration of pyridoxine 24hr. before the rats were killed reversed the changes in incorporation of radioactivity from [¹⁴C]formate. 3. In pyridoxine deficiency, the incorporation of radioactivity from dl-[3-¹⁴C]serine, [8-¹⁴C]adenine, [Me-³H]thymidine and [2-¹⁴C]deoxyuridine was decreased. The incorporation of radioactivity from l-[Me-¹⁴C]methionine was not affected. No noteworthy differences in the effect of pyridoxine deficiency on the incorporation of radioactivity from dl-[3-¹⁴C]serine into DNA and RNA were observed, whereas the effect of the deficiency on the incorporation of radioactivity from [8-¹⁴C]adenine into spleen DNA was somewhat greater than that into spleen RNA. Administration of pyridoxine 24hr. before the rats were killed reversed the changes in incorporation of radioactivity from [3-¹⁴C]serine and [8-¹⁴C]adenine. 4. The adverse effects of pyridoxine deficiency on the biosynthesis of nucleic acids and cell multiplication are discussed in relation to the role of pyridoxal phosphate in the production of C₁ units via the serine-hydroxymethylase reaction.     

The action of overdosed biotin on reproduction of the hide beetle,Dermestes maculatus

Excessive biotin (cis-tetrahydro-2-oxothieno[3,4-d]-imidazoline-4-valeric acid) intake by female Dermestes maculatus permits protein incorporation into yolk but suppresses embryogenesis during its later stage, presumably due to partial inactivation of egg proteases. Experiments with dietary biotin-carbonyl-¹⁴C suggested that the overdosed vitamin forms a complex with insoluble yolk proteins. The superfluous vitamin does not curtail the activity of acid phosphatase in young embryos. Hide beetles, sterilized by 1·0% dietary biotin, incorporate to their eggs about 2·3 times more biotin than control females, whereas the former excrete about 27·4 times more biotin than the latter. The adults seem to eliminate the vitamin surplus less efficiently than the larvae. The significance of impaired utilization of yolk proteins is discussed as a means for insect chemosterilization.     

A possible mechanism of inhibition of protein synthesis by dimethylnitrosamine

1. The incorporation of [¹⁴C]leucine into liver proteins of rats was measured in vivo at various times after treatment of the animals with dimethylnitrosamine and was correlated with the state of the liver ribosomal aggregates. Inhibition of incorporation ran parallel with breakdown of the aggregates. 2. Inhibition of leucine incorporation into protein and breakdown of ribosomal aggregates were not preceded by inhibition of incorporation of [¹⁴C]orotate into nuclear RNA of the liver. 3. Evidence was obtained of methylation of nuclear RNA in the livers of rats treated with [¹⁴C]dimethylnitrosamine. 4. Zonal centrifugation analysis of radioactive, nuclear, ribosomal and transfer RNA from livers of rats treated with [¹⁴C]dimethylnitrosamine revealed labelling of all centrifugal fractions to about the same extent. 5. It is suggested that methylation of messenger RNA might occur in the livers of dimethylnitrosamine-treated rats and the possible relation of this to inhibition of hepatic protein synthesis is discussed.     

In Vitro Incorporation of Selenomethionine into Protein by Vigna radiata Polysomes

Vigna radiata polysomes efficiently incorporated [⁷⁵Se]selenomethionine, [¹⁴C]methionine, and [¹⁴C]leucine in vitro. The optimal conditions for translation were determined to be 4.8 millimolar Mg²⁺, 182 millimolar K⁺, and pH 7.4. The rates of incorporation of [⁷⁵Se]selenomethionine and [¹⁴C]methionine were similar when measured separately, but [⁷⁵Se]selenomethionine incorporation was 35% less than [¹⁴C]methionine incorporation when both amino acids were present in equal molar concentrations. Polyacrylamide gel electrophoresis of the hot trichloroacetic acid precipitable translation products demonstrated synthesis of high molecular weight labeled proteins in the presence of [⁷⁵Se]selenomethionine or [³⁵S]methionine. No major differences in molecular weights could be detected in the electrophoretic profiles. Utilization of selenomethionine during translation by Vigna radiata polysomes establishes a route for the assimilation of selenomethionine by plants susceptible to selenium toxicity.     

Bacitracin Inhibits the Synthesis of Lipid-linked Saccharides and Glycoproteins in Plants

The particulate enzyme fraction from mung bean (Phaseolus aureus) seedlings catalyzes the incorporation of mannose from GDP-[¹⁴C]mannose into mannosyl-phosphoryl-dolichol and of N-acetylglucosamine from UDP-[³H]N-acetylglucosamine into N-acetylglucosamine-pyrophosphoryl-polyisoprenol. Bacitracin inhibits the transfer of both of these sugars into the lipid-linked saccharides with 50% inhibition being observed at 5 mm bacitracin. This antibiotic did not inhibit the transfer of glucose from UDP-[¹⁴C]glucose into steryl glucosides or the incorporation of glucose into a cell wall glucan. Bacitracin also inhibited the in vivo incorporation of [¹⁴C]mannose into mannosyl-phosphoryl-dolichol and into glycoprotein by carrot (Daucus carota) slices. While bacitracin also inhibited the incorporation of lysine into proteins by these slices, protein synthesis was less sensitive than glycosylation. Thus at 2 mm bacitracin glycosylation was inhibited 92%, while protein synthesis was inhibited only 50%.     

Coronatine biosynthesis: Incorporation of l-[U-14C]isoleucine and l-[U-14C] threonine into the 1-amido-1-carboxy-2-ethylcyclopropyl moiety

Addition of either l-[U-¹⁴C]threonine or l-[U-¹⁴C]isoleucine to 2.7-day-old shaking liquid cultures of Pseudomonas syringae pv. atropurpurea resulted in incorporation of radioactivity into coronatine, but not into N- coronafacoylvaline, another phytotoxin excreted by P.s. atropurpurea. In contrast, addition ofl-[U-¹⁴C]valine did not lead to incorporation of radioactivity into coronatine, but instead into coronafacoylvaline. Acid hydrolysis of the purified [¹⁴C] coronatine obtained after incorporation of either [¹⁴C]isoleucine or [¹⁴C]threonine demonstrated that > 94% of the radioactivity was present in the 1-amido-1-carboxy-2-ethylcyclopropyl moiety of coronatine, and < 6 % was in the coronafacoyl moiety. These findings are used to propose a biosynthetic pathway for coronatine.     

Host-Pathogen Interactions : XXIV. Fragments Isolated from Suspension-Cultured Sycamore Cell Walls Inhibit the Ability of the Cells to Incorporate [C]Leucine into Proteins

A bioassay to measure the incorporation of [¹⁴C]leucine into acid-precipitable polymers of suspension-cultured sycamore (Acer pseudoplatanus L.) cells is described. Using this assay, cell wall fragments solubilized from sycamore cell walls by partial acid hydrolysis are shown to contain components that inhibit the incorporation of [¹⁴C]leucine into the acid-precipitable polymers. This inhibition was not attributable to a suppression of [¹⁴C]leucine uptake. The effectiveness of the wall fragments in inhibiting [¹⁴C]leucine incorporation was substantially relieved by plasmolysis of the cells. Fragments released from starch and citrus pectin are shown not to possess such inhibitory activities.     

The effect of hypolipidemic drugs WY14643 and DH990, and lysophospholipids on the metabolism of oleate in plants

The effects of the addition of hypolipidemic drugs and 1-acylglycerolipids on the metabolism of oleate in plants have been studied in vivo and in vitro. Using aged potato slices with [¹⁴C]oleate as a precursor, it was found that these drugs markedly inhibited both the incorporation into complex lipids and the desaturation of oleate to linoleate. Moreover, in vitro experiments, carried out with microsomes prepared from developing safflower seeds and [¹⁴C]oleate or [¹⁴C]oleoyl-CoA as precursors, confirmed the inhibitory effect of the drugs on oleate desaturation, and showed that while WY14643 mainly affected oleoyl thiokinase activity, DH990 exerted its strongest effect on the formation of PL, indicating that the mode of action of these two drugs in safflower microsomes is essentially different. Addition of LPC or LPE stimulated the incorporation of radiolabeled precursor into PC and PE, respectively, as well as the desaturation of oleate to linoleate when [¹⁴C]oleoyl-CoA was the precursor. The evidence obtained suggests that oleoyl-PE, as well as oleoyl-PC, should be considered as a possible substrate for oleate desaturation in plants.     

Fluoride-Induced Inhibition of Starch Biosynthesis in Developing Potato, Solanum tuberosum L., Tubers Is Associated with Pyrophosphate Accumulation

Pretreatment of discs excised from developing tubers of potato (Solanum tuberosum L.) with 10 millimolar sodium fluoride induced a transient increase in 3-phosphoglycerate content. This was followed by increases in triose-phosphate, fructose 1,6-bisphosphate and hexose-phosphate (glucose 6-phosphate + fructose 6-phosphate + glucose 1-phosphate). The effect of fluoride is attributed to an inhibition of glycolysis and a stimulation of triose-phosphate recycling (the latter confirmed by the pattern of ¹³C-labeling [NMR] in sucrose when tissue was supplied with [2-¹³C]glucose). Fluoride inhibited the incorporation of [U-¹⁴C] glucose, [U-¹⁴C]sucrose, [U-¹⁴C]glucose 1-phosphate, and [U-¹⁴C] glycerol into starch. The incorporation of [U-¹⁴C]ADPglucose was unaffected. Inhibition of starch biosynthesis was accompanied by an almost proportional increase in the incorporation of ¹⁴C into sucrose. The inhibition of starch synthesis was accompanied by a 10-fold increase in tissue pyrophosphate (PPi) content. Although the subcellular localization of PPi was not determined, a hypothesis is presented that argues that the PPi accumulates in the amyloplast due to inhibition of alkaline inorganic pyrophosphatase by fluoride ions.     

Metabolism of nicotine in Nicotiana glauca

A mixture of (?)-nicotine-[2′-³H] and (±)-nicotine-[2′-¹⁴C] was administered to Nicotiana glauca plants for 3 days, resulting in the formation of radioactive nornicotine (49·5% incorporation) and myosmine (2·05% incorporation). Negligible activity was detected in anabasine, cotinine, or 3-acetylpyridine, the last two compounds being added as carriers to the harvested plants. The radioactive nornicotine consisted of 48% (?)-nornicotine-[2′-¹⁴C,³H] and 52% (+)-nornicotine-[2′-¹⁴C]. Thus if (+)-nornicotine is formed from (?)-nicotine the transformation must involve loss of the hydrogen from C-2′. Myosmine is presumably formed from nicotine via nornicotine. However by feeding myosmine-[2′-¹⁴C] to N. glauca it was shown that the dehydrogenation is not reversible, no activity being detected in nornicotine. Nicotinic acid (0·14% incorporation) was a metabolite of myosmine-[2′-¹⁴C]. Essentially all the activity of the nicotinic acid was located on its carboxyl group, indicating that myosmine was a direct precursor.     

THE METABOLISM OF LEUCINE BY DEVELOPING RAT BRAIN: EFFECT OF LEUCINE AND 2-OXO-4-METHYLVALERATE ON LIPID SYNTHESIS FROM GLUCOSE AND KETONE BODIES

Abstract— The oxidation of l -[U-¹⁴C]leucine and l -[l-¹⁴C]leucine at varying concentrations from 0.1 to 5mM to CO₂ and the incorporation into cerebral lipids and proteins by brain slices from 1-week old rats were markedly stimulated by glucose. Although the addition of S mM-dl -3-hydroxybutyrate had no effect on the metabolism of [U-¹⁴C]leucine by brain slices from suckling rats, the stimulatory effects of glucose on the metabolism of l -[U-¹⁴C]leucine were markedly reduced in the presence of dl -3-hydroxybutyrate. The stimulatory effect of glucose on leucine oxidation was, however, not observed in adult rat brain. Furthermore, the incorporation of leucine-carbon into cerebral lipids and proteins was also very low in the adult brain. The incorporation of l -[U-¹⁴C]leucine into cerebral lipids by cortex slices was higher during the first 2 postnatal weeks, which then declined to the adult level. During this time span, the oxidation of l -[U-¹⁴C]leucine to CO₂ remained relatively unchanged. The incorporation in vivo of D-3-hydroxy[3-¹⁴C]butyrate into cerebral lipids was markedly decreased by acute hyperleucinemia induced by injecting leucine into 9-day old rats. In in vitro experiments, 5 mM-leucine had no effect on the oxidation of [U-¹⁴C]glucose to CO₂ or its incorporation into lipids by brain slices from 1-week old rats. However, 5 mM-leucine inhibited the oxidation of d -3-hydroxy-[3-¹⁴C]butyrate, [3-¹⁴C]acetoacetate and [1-¹⁴C]acetate to CO₂ by brain slices, but their incorporation into cerebral lipids was not affected by leucine. In contrast 2-oxo-4-methylvalerate, a deaminated metabolite of leucine, markedly inhibited both the oxidation to CO₂ and the incorporation into lipids of labelled glucose, ketone bodies and acetate by cortex slices from 1-week old rats. These findings suggest that the reduction in the incorporation in vivo of d -3-hydroxy[3-¹⁴C]butyrate into cerebral lipids in rats injected with leucine is most likely caused by 2-oxo-4-methylvalerate formed from leucine. Since the concentrations of leucine and 2-oxo-4-methylvalerate in plasma of untreated patients with maple-syrup urine disease are markedly elevated, our findings are compatible with the possibility that an alteration in the metabolism of glucose and ketone bodies in the brain may contribute to the pathophysiology of this disease.