Biosynthesis of C-labeled branched polysaccharides by pestalotiopsis from C-labeled glucoses and the mechanism of formation

Biosynthesis of branched glucan by Pestalotiopsis from media containing D-(1-¹³C)glucose, D-(2-¹³C)glucose, D-(4-¹³C)glucose, D-(6-¹³C)glucose or a mixture of D-(1-¹³C)glucose and D-(2-¹³C)glucose was carried out to elucidate biosynthetic mechanism of branched polysaccharides. ¹³C NMR spectra of the labeled polysaccharides were determined and assigned. Analysis of ¹³C NMR spectra of glucitol acetates obtained from hydrolysates of the labeled branched polysaccharides indicated that transfer of labeling from C-1 to C-3 and C-6 carbons, from C-2 to C-1, C-3 and C-5 carbons, and from C-6 to C-1 carbon. From the results the percentages of routes via which the polysaccharide is biosynthesized are estimated. They show that the biosynthesis of the polysaccharide via the Embden-Meyerhof pathway and that from lipids and proteins are more active, and the pentose cycle is less active, than in the biosynthesis of cellulose and curdlan. As for the results, labeling at C-6 carbon in the branched polysaccharide cultured from D-(6-¹³C)glucose was low, compared to that of cellulose and curdlan.     

Biosynthetic studies of lactucin derivatives in hairy root cultures of Lactuca floridana

Biosynthetic studies of the guaianolide-type sesquiterpene lactones 11βH,13-dihydrolactucin-8-O-acetate and 8-desoxylactucin were performed in Agrobacterium rhizogenes—transformed hairy root cultures of blue-flowered lettuce, Lactuca floridana. The ¹³C NMR spectra of the two guaianolides labelled by incorporation of [1-¹³C], [2-¹³C], [1,2-¹³C₂]acetate and [2-¹³C]mevalolactone showed patterns of enrichment consistent with a previously proposed biogenetic pathway for guaianolide-type sesquiterpene lactones via the acetate-mevalonate-germacradiene route.     

Regulation of pathways of glucose metabolism in kidney. The effect of experimental diabetes on the activity of the pentose phosphate pathway and the glucuronate-xylulose pathway.

Measurements have been made of the activities of enzymes of the pentose phosphate pathway, the glucuronate-xylulose pathway, hexokinase and phosphofructokinase in kidney of diabetic and normal rats. The activities of these enzymes keep pace with kidney growth, remaining constant per gram tissue but showing a marked increase on the basis of total activity per 100 g body wt. The formation of ¹⁴CO₂ from [1-¹⁴C]glucose and [6-¹⁴C]glucose by kidney slices from diabetic rats was decreased to approximately half the control value; evidence was obtained for an equivalent dilution of the glucose 6-phosphate pool. Correction of the ¹⁴CO₂ yields for the change in specific activity of glucose 6-phosphate yielded values consistent with the enzyme profile. Calculations from specific yields of ¹⁴CO₂ provided evidence for an increased flux of glucose via the pentose phosphate pathway in the kidney in diabetes. The results are discussed in relation to kidney hypertrophy in diabetes and the requirement for ribose 5-phosphate and NADPH for biosynthetic reactions and in relation to the thickening of the basement membrane in diabetes. These results are in accord with the concept of glucose overutilization by non-insulin-requiring tissues.     

The sequence of initiation of RNA, DNA and protein synthesis in the wheat grains during germination

The syntheses of main macromolecular substances, in a whole wheat grain allowed to germinate, are triggered in the following order: RNA, protein, DNA. The RNA synthesis, as judged by [2-¹⁴C]uridine incorporation, is initiated almost immediately after the seeds are exposed to the optimal germination conditions, whereas [1-¹⁴C]leucine and [2-¹⁴C]thymidine incorporation begins to occur only 3 and 4 hr later, respectively. The initiation of protein synthesis is accompanied by an apparent cessation of uridine incorporation.     

Oxytocin and glucose oxidation in the rat uterus

The effects of oxytocin on the biochemical pathways of glucose oxidation were investigated in the rat uterus. In the presence of oxytocin, glucose oxidation in uterine segments obtained from Sprague-Dawley rats at diestrus increased 1.5–2.0-fold above the basal rate. A half-maximal response was observed at about 3 nM oxytocin; the maximum response was equal to or greater than the response to 1.7 nM insulin. In stripped myometrial segments (denuded of the endometrial component), oxytocin stimulated glucose oxidation at estrus only; whereas in intact uterine segments, the stimulation of oxidation was observed at both estrus and diestrus. In contrast, stimulation of oxidation by carbachol in stripped myometrial segments was independent of the estrous state of the tissue. The ratio of [1-¹⁴C]glucose to [6-¹⁴C]glucose oxidation was measured to estimate the relative involvement of the pentose phosphate and the tricarboxylic acid pathways of metabolism. In myometrial tissue, stimulation of glucose oxidation by oxytocin appeared to proceed through the tricarboxylic acid cycle. In intact uterine segments, at diestrus, glucose oxidation involved largely the pentose phosphate pathway (suggesting increased glucose metabolism in endometrial tissue), whereas at estrus, in the intact tissue segments, oxytocin increased glucose oxidation largely via the tricarboxylic acid cycle, and appeared to do so predominantly in the myometrial tissue. Carbachol-stimulated glucose oxidation appeared to proceed mainly via the tricarboxylic cycle in the myometrial tissue, irrespective of the stage of the estrous cycle. In the uterus of the Brattleboro rat (either intact uterine segments or stripped myometrial strips), oxytocin stimulated glucose oxidation only at estrus, predominantly through the tricarboxylic acid cycle. These findings suggest that oxytocin, in addition to its known effect on the contractility of uterine and myoepithelial smooth muscle, may regulate glucose metabolism in both the myometrial and endometrial components of uterine tissue.     

TIME-DEPENDENT PARTITIONING OF GLUCOSE CARBONS METABOLIZED BY THE SUPERIOR CERVICAL GANGLION FROM CALVES

Abstract— Glucose metabolism in the superior cervical ganglion for calves has been studied by incubating slices with [1-¹⁴C]-, [6-¹⁴C]- and [U-¹⁴C]-labelled glucose at 37°C and pH 7.4. Glucose utilization and the metabolic partitioning of glucose carbon in products during different incubation periods ranging from 5 to 60 min were determined by isotopic methods.
Separation and identification of labelled compounds have been achieved by anion and cation exchange chromatography as well as by TLC and enzymatic analyses.
From the data obtained a carbon balance could be constructed showing lactate to be the major product of glucose metabolism followed by CO₂ and amino acids. Measuring the release of ¹⁴CO₂ from differently ⁴C-labelled glucose, the existence of an active pentose phosphate pathway in the ganglion could be demonstrated although this pathway seems to contribute only to a small extent to glucose metabolism. The marked decrease of the C-U: C-6 and the C-U:C-1 ratios in ¹⁴CO₂ observed in the course of incubation is discussed in terms of a time-dependent change in the rate of synthesis of amino acids which are directly connected with intermediates of the citric acid cycle.     

Inhibitory effect of inorganic phosphate on the axonemal ATPase of cilia from Tetrahymena pyriformis

An inhibitory effect of inorganic phosphate on the axonemal ATPase of cilia from Tetrahymena pyriformis was shown. P_i inhibited the terminal phosphate liberation from [γ-³²P]ATP by 30-S dynein and inhibited the conversion of [8-¹⁴C]ATP to ADP and AMP by digitonin-extracted cilia.     

Modulation of protein kinase C activity by NaF in bone marrow derived macrophages

Stimulation of murine bone marrow derived macrophages with NaF, prelabeled with [1-¹⁴C]oleate and [³H]inositol, increased the production of inositol phosphates and the release of 1,2-[¹⁴C]diacylglycerol (DAG). Moreover, NaF also induced activation of protein kinase C. These results indicate that bone marrow derived macrophages exhibit a phosphatidyl-4,5-bisphosphate phospholipase C activity, sensitive to NaF, which might be modulated by G-proteins. Activation of protein kinase C could have been mediated by NaF-induced release of DAG.     

Bradykinin effects on phospholipid metabolism and its relation to arachidonic acid turnovers in neuroblastoma × glioma hybrid cells (NG 108-15)

In neuroblastoma × glioma hybrid cells (NG 108-15) labelled with [³²P]-trisodium phosphate, [³H]-inositol and [¹⁴C]-arachidonic acid, bradykinin stimulated the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) while it had no effect on the release of [¹⁴C]-arachidonic acid (AA). The effect on PIP, was time- and dose-dependent with a maximal effect on [³H]-inositol- and [³²P]-labelled cells after 10–30 s of stimulation with 10⁻⁶ M bradykinin. However, the hydrolysis of [¹⁴C]-AA labelled PIP₂ was delayed compared to the effect on [³H]- and [¹⁴C]-PIP₂ and was not detectable until after 60 s of stimulation. Bradykinin stimulation resulted in an increased formation of [³H]-inositol phosphates (IP) and [³²P]- and [¹⁴P]- and [¹⁴C]-phosphatidic acid (PA) but the time course for PA formation did not allow the time-course for PIP₂ hydrolysis. A reduced labelling of [²³P]- and [¹⁴C]-phosphatidylcholine was also found in stimulated cells suggesting that PA may derive from other sources than PIP₂. In conclusion, our results indicate that bradykinin activates phospholipase C, but not phospholipase A₂, in NG 108-15 cells.     

Differential production of prostaglandins D2 and E2 and of unusual prostanoid by chick spinal cord and meninges in vitro

In order to specify the source of locally synthesized prostaglandin (PG) E₂ which is able to saturate the large class of low affinity PGE₂ receptors in chick spinal cord, bioconversion of [1-¹⁴C]arachidonic acid into prostanoids was studied in homogenates of chick spinal cord and meninges first without addition of exogenous glutathione (GSH). Homogenates of spinal cord produced ¹⁴C-labeled PGE₂, PGD₂ and PGF₂. Homogenates of meninges accumulated much larger amounts of [¹⁴C]PGE₂ than spinal cord and surprisingly a ¹⁴C-labeled arachidonate metabolite referred to as compound Y. Compound Y generation, which was inhibited by indomethacin and enhanced by esculetin, was therefore mediated through the cyclooxygenase pathway. The fact that no labeled compound Y was detected in homogenates incubated with [³H]PGD₂ or [³H]PGE₂ indicated that compound Y was not degradation product of PG_s. Secondly, after addition of exogenous GSH, ¹⁴C-labeled compound Y was totally converted into [¹⁴C]PGE₂. The compound Y which is converted into PGF_s after a strong reduction with NaBH₄ and into PGE₂ after a mild reduction with GSH-hemin system or SnCl₂ was therefore assumed to be a 15 hydroperoxy-PGE₂ (15 HP-PGE₂). These results suggest that PGE₂ can be synthesized in meninges either by the classical isomerization of PGH₂ or by isomerization of PGG₂ followed by a GSH-sensitive reaction.     

Ascorbic acid catabolism by gut microflora: studies in germ-free and conventional guinea pigs

The role of gut microflora in ascorbic acid catabolism was investigated in both conventional and germ-free guinea pigs. In vitro studies demonstrated extensive degradation of the vitamin by fresh feces, cecal, and colonic contents of conventional guinea pigs. Direct injection of [1-¹⁴C] ascorbic acid into the cecum of conventional guinea pigs in vivo yielded a 70% recovery of the label as respiratory ¹⁴CO₂ within 6 hr compared with only 5% recovery following injection into the virtually sterile peritoneum in a comparable group of conventional guinea pigs. Thus, ascorbic acid not absorbed prior to reaching the lower gastrointestinal tract stands to be extensively decarboxylated by microflora in the cecum. In a companion study of germ-free guinea pigs, 10% of an administered dose of [1-¹⁴C] ascorbic acid was expired as ¹⁴CO₂ within 36 hr post-injection following intraperitoneal injection compared with 16% recovery in a matched group of conventional animals injected at the same site. Results of this series of studies suggest that hepatic decarboxylation and gut microflora, in tandem, contribute to ascorbic acid decarboxylation in this species.     

Quantitative measurement of the L-type pentose phosphate cycle with [2-14C]glucose and [5-14C]glucose in isolated hepatocytes.

1. Investigations of the mechanism of the non-oxidative segment of the pentose phosphate cycle in isolatd hepatocytes by prediction-labelling studies following the metabolism of [2-14C]-, [5-14C]- and [4,5,6-14C]glucose are reported. The 14C distribution patterns in glucose 6-phosphate show that the reactions of the L-type pentose pathway in hepatocytes. 2. Estimates of the quantitative contribution of the L-type pentose cycle are the exclusive form of the pentose cycle to glucose metabolism have been made. The contribution of the L-type pentose cycle to the metabolism of glucose lies between 22 and 30% in isolated hepatocytes. 3. The distribution of 14C in the carbon atoms of glucose 6-phosphate following the metabolism of [4,5,6-14C]- and [2-14C]glucose indicate that gluconeogenesis from triose phosphate and non-oxidative formation of pentose 5-phosphate do not contribute significantly to randomization of 14C in isolated hepatocytes. The transaldolase exchange reaction between fructose 6-phosphate and glyceraldehyde 3-phosphate is very active in these cells.     

The effect of exogenous gibberellic acid on gibberellin biosynthesis by Gibberella fujikuroi

The addition of gibberellic acid and some other gibberellins to cultures of Gibberella fujikuroi suppresses the incorporation of [2-¹⁴C]MVA and ¹⁴C-labelled ent-kaurene into the gibberellin metabolises.     

Metabolism of some possible ecdysone precursors in Calliphora stygia

A study has been made, using Calliphora stygia at the time of puparium formation, of the incorporation of a number of labelled sterols into β-ecdysone. [1-³H]-Cholesterol and [4-¹⁴C]-cholesterol are incorporated to a similar extent (0·01-0·02%). [1-³H]-7-Dehydrocholesterol is better incorporated (0·025%) than cholesterol while [1-³H]-cholesterol sulphate, (22R)-22-hydroxy-[22-³H]-cholesterol, and 25-hydroxy-[26-¹⁴C]-cholesterol are not incorporated to a significant extent.     

Extraglandular hormonal steroidogenesis in aged rats

We have examined the metabolism in vitro of [4-¹⁴C]pregnenolone by the following organs of 2.4-year-old rats: submandibular gland, stomach, duodenum, liver, lung, heart, spleen, kidney, skin, prostate, testis and adrenal. All tissues converted pregnenolone to progesterone, the highest yields being observed with adrenal, testis and skin. Androgen formation was intense in the testis and absent in the adrenal. Moreover, 17-hydroxylation of pregnenolone occurred moderately in kidney, skin and submandibular gland and markedly in duodenum and stomach, which also produced high amounts of dehydroepiandrosterone and/or 5-androstene-3β,17β-diol. Extratesticular synthesis of androstenedione and testosterone was very low. A significant formation of 20-dihydropregnenolone was observed in all tissues but stomach, duodenum and steroidogenic endocrines. Corticosteroids were not synthesized extraadrenally, except a small amount of 11-deoxycorticosterone in the testis. These results indicate that key steroid-biosynthetic enzymes, such as 3β-hydroxysteroid dehydrogenase/Δ⁵′Δ⁴ isomerase, 17β- and 20-hydroxysteroid dehydrogenases and steroid 17-monooxygenase/17,20-lyase are also expressed extraglandularly in the rat.     

Analysis of the biosynthetic process of cellulose and curdlan using C-labeled glucoses

In order to elucidate the biosynthetic process of cellulose and curdlan, ¹³C-labeled polysaccharides were biosynthesized by Acetobacter xylinum (IFO 13693) and Agrobacterium sp. (ATCC 31749), from culture media containing -(1-¹³C)glucose, -(2-¹³C)glucose, -(4-¹³C)glucose, or -(6-¹³C)glucose as the carbon source, and their structures were determined by ¹³C NMR spectroscopy. The labeling was mainly found in the original position, indicating direct polymerization of introduced glucoses. In addition, the transfer of labeling from C-2 to C-1, C-3 and C-5, from C-4 to C-1, C-2 and C-3, and from C-6 to C-1 was found in celluloses. In curdlan, the transfer of labeling from C-1 to C-3, from C-2 to C-1 and C-3, from C-4 to C-1, C-2 and C-3, and from C-6 to C-1 and C-3 was observed. From analysis of this labeling, the biosynthetic process of cellulose and curdlan was explained as involving six routes. The percentages of each route via which cellulose or curdlan is biosynthesized were estimated for upper (C-1 to C-3) and lower portions (C-4 to C-6) of glucosidic units in the polysaccharides. It is noted that very few polysaccharides are formed via the Embden-Meyerhof pathway. The lower half (C-4 to C-6) structure of introduced glucoses is well preserved in the polysaccharides.     

On the mechanism of biosynthesis of leukotrienes and related compounds

[10D-³H; 3-¹⁴C]- and [10L-³H; 3-¹⁴C]arachidonic acids were incubated with human polymorphonuclear leukocytes and with human platelets. Leukotriene B₄ and 5(S),12(S)-dihydroxy-6trans,8cis,10trans,14-cis-eicosatetraenoic acid (5,12-DHETE) were isolated and the ³H/¹⁴C ratios determined. It could be concluded that the 10D (pro-R)-hydrogen is eliminated in the conversion of 5(S)-hydroperoxy-6trans,8cis,11cis,14cis-eicosatetraenoic acid into leukotriene A₄ whereas in the conversion of arachidonic acid into 5,12-DHETE the 10L (pro-S)-hydrogen is lost. Incubation of the doubly labeled arachidonic acids with human platelets confirmed and extended previous data on the stereochemistry of the hydrogen removal from C-10 during the conversion into 12(S)-hydroperoxy-5cis,8cis,10trans,14cis-eicosatetraenoic acid, i.e., the 10L (pro-S)-hydrogen is eliminated and the 10D (pro-R)-hydrogen retained.     

The pentylenetetrazole-kindling model of epilepsy in SAMP8 mice: glial-neuronal metabolic interactions

Recently, a new experimental model of epilepsy was introduced by the authors [Neurochem. Int. 40 (2002) 413]. This model combines pentylenetetrazole (PTZ)-kindling in senescence-accelerated mice P8 (SAMP8), a genetic model of aging. Since imbalance of glutamate and GABA is a major cause of seizures, the study of glial–neuronal interactions is of primary importance. Nuclear magnetic resonance spectroscopy (NMRS) is an excellent tool for metabolic studies. Thus, we examined whether NMRS when combined with administration of [1-¹³C]glucose and [1,2-¹³C]acetate might give valuable insights into neurotransmitter metabolism in this new model of epilepsy and aging. The 2- and 8-month-old SAMP8 were kindled with PTZ alone, received PTZ and phenobarbital (PB), or served as controls. In older animals, PTZ-kindling decreased labeling in glutamate C-4 from [1-¹³C]glucose, whereas, in the younger mice, labeling in glutamine C-4 was decreased both from [1-¹³C]glucose and [1,2-¹³C]acetate. It could be concluded that PTZ-kindling affected astrocytes in younger and glutamatergic neurons in older animals. In the presence of PTZ, phenobarbital decreased labeling of most metabolites in all cell types, except GABAergic neurons, from both labeled precursors in the younger animals. However, in older animals only GABAergic neurons were affected by phenobarbital as indicated by an increase in GABA labeling.     

Effects of dietary paraffin, squalane and sucrose polyester on residue disposition and elimination of hexachlorobenzene in rats

Previous studies have shown addition of light liquid paraffin to enhance the elimination of organochlorine xenobiotics. In the present study the effect of paraffin on the elimination of [¹⁴C]hexachlorobenzene (HCB) was compared with the effect of possible alternative compounds, squalane and sucrose polyester (SPE). Four groups of 7 rats were fed a diet containing 1.5 ppm [¹⁴C]HCB for 4 days followed by 10 days on HCB-free diet. Thereafter one group (control) remained on this diet whereas the other 3 groups received a diet supplemented with 8% (w/w) paraffin, squalane or SPE, respectively. Radioactivity in urine and faeces was measured daily and at the end of the experiment in samples of abdominal fat, muscle, liver, kidney and blood. Dietary treatment with either paraffin, squalane or SPE markedly enhanced faecal excretion of [¹⁴C]HCB, whereas urinary excretion was not affected. Both the time course as well as the extent of faecal [¹⁴C]HCB elimination were similar in the treated groups. After 3 weeks of treatment the amount of [¹⁴C]HCB excreted with faeces was about three times higher in treated animals than in controls. The half-life (t_1/2) of [¹⁴C]HCB elimination from the body was markedly decreased in treated animals (mean 34–38 days) compared to controls (110 days). [¹⁴C]HCB concentrations in some major tissues were significantly reduced to the same extent by all three dietary regimens. Thus squalane and SPE are as effective as paraffin in removing HCB from contaminated animals.     

Glucose fermentation to acetate and alanine in resting cell suspensions of Pyrococcus furiosus: Proposal of a novel glycolytic pathway based on 13C labelling data and enzyme activities

Abstract Suspensions of maltose-grown cells of the hyperthermophilic archaeon Pyrococcus furiosus , when incubated at 90°C with 35 mM [1-¹³C]glucose or [3-¹³C]glucose, consumed glucose at a rate of about 10 nmol min⁻¹ (mg protein)⁻¹. Acetate (10 mM), alanine (3 mM), CO₂ and H₂ were the fermentation products. The ¹³C-labelling pattern in alamine and acetate were analyzed. With [1-¹³C]glucose the methyl group of both alanine and acetate was labelled; with [3-¹³C]glucose only the carboxyl group of alanine was labelled whereas acetate was unlabelled. Extracts of maltose-grown cells contained glucose isomerase (12.8 U mg⁻¹, 100°C), ketohexokinase (0.23 U mg⁻¹, 100°C), and fructose 1-phosphate aldolase (0.06 U mg^{−1, 100°C). Enzymes catalyzing the formation of fructose 1,6-bisphosphate from fructose 1-phosphate or fructose 6-phosphate could not be detected. As publihed previously by our group and other authors P. furiosus also contains enzymes of glyceraldehyde conversion to 2-phosphoglycerate according to a non-phosphorylated Entner-Doudoroff pathway, of dihydroxyacetone phosphate conversion to 2-phosphoglycerate according to the Embden-Meyerhof pathway, and of 2-phosphoglycerate conversion - via pyruvate - to acetate and alanine. Based on the enzyme activities in P. furiosus , the following pathway for glucose degradation to alanine and acetate in cell suspensions is proposed which can explain the [13}C]glucose labelling data: glucose→ fructose → fructose 1- phosphate → dihydroxyacetone phosphate + glyceraldehyde and further conversion of both trioses to alanine and acetate via pyruvate.