Biomedical subjects

A Lapidot

Publications and source records attributed to A Lapidot.

At least 37 records · Page 2Linked to original sources

PubMed1994

Cerebral metabolic compartmentation. Estimation of glucose flux via pyruvate carboxylase/pyruvate dehydrogenase by 13C NMR isotopomer analysis of D-[U-13C]glucose metabolites.

A method is presented for determining the compartmentation of amino acid metabolism in the brain. 13C NMR spectroscopy, and more specifically, homonuclear 13C-13C spin coupling patterns of 13C-labeled amino acids were used to measure the relative flux of label from D-[U-13C]glucose through the anaplerotic pathway versus the oxidative pathway. Glucose flux through the pyruvate carboxylase pathway was quantitated following primed dose constant infusion of D-[U-13C]glucose to young rabbits at a rate of 1 mg/kg body weight per min. We demonstrate, for the first time, that multiplet spectra of three adjacent 13C isotopomer in 1,2,3-13C3 in glutamine and glutamate, which are derived from [1,2,3-13C3]pyruvate, present different isotopomer populations in glutamine in comparison to that in glutamate. This is due to two different metabolic compartments characterized by the presence or absence of glutamine synthetase activity and two different tricarboxylic acid cycles, one preferentially mediated by pyruvate carboxylase and the other by pyruvate dehydrogenase. Our results indicate that the anaplerotic pathway accounts for 34% of glutamine synthesis and only 16% of glutamate and gamma-aminobutyric acid syntheses in metabolic and isotopic steady state conditions. These results support the concept, and provide a quantitative measure, that glutamine and/or tricarboxylic acid cycle intermediates are supplied by astrocytes to neurons to replenish the neurotransmitter pool of gamma-aminobutyric acid and glutamate.

Animals↗

PubMed1994

Cloning and DNA sequence of the gene coding for Bacillus stearothermophilus T-6 xylanase.

Bacillus stearothermophilus T-6 produces an extracellular thermostable xylanase. Affinity-purified polyclonal serum raised against the enzyme was used to screen a genomic library of B. stearothermophilus T-6 constructed in lambda-EMBL3. Two positive phages were isolated, both containing similar 13-kb inserts, and their lysates exhibited xylanase activity. A 3,696-bp SalI-BamHI fragment containing the xylanase gene was subcloned in Escherichia coli and subsequently sequenced. The open reading frame of xylanase T-6 consists of 1,236 bp. On the basis of sequence similarity, two possible -10 and -35 regions, a ribosome-binding site at the 5' end of the gene and a potential transcriptional termination motif at the 3' end of the gene, were identified. From the previously known N-terminal amino acid sequence of xylanase T-6 and the possible ribosome-binding site, a putative 28-amino-acid signal peptide was deduced. The mature xylanase T-6 consists of 379 amino acids with a calculated molecular weight and pI of 43,808 and 6.88, respectively. Multiple alignment of beta-glycanase amino acid sequences revealed highly conserved regions. Northern (RNA) blot analysis indicated that the xylanase T-6 transcript is about 1.4 kb and that the induction of this enzyme synthesis by xylose is on the transcriptional level.

Amino Acid Sequence↗

PubMed1993

The conformation of new tetrahydropyrimidine derivatives in solution and in the crystal.

We have recently identified by NMR techniques two new tetrahydropyrimidine derivatives, 2-methyl-4-carboxy-5-hydroxy-3,4,5,6-tetrahydropyrimidine [THP(A)] and 2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine [THP(B)], which accumulated in actinomycin D producing Streptomyces parvulus. Their relatively high intracellular concentrations during actinomycin D synthesis and identity of the time of onset of their synthesis with that of actinomycin D synthesis leads us to suggest that they may function in the self-defense mechanism of actinomycin-producing organisms. Here we present a combination of one-dimensional and two-dimensional 1H- and 13C-NMR studies in solution and X-ray crystallography of THP(A) and THP(B). Our results demonstrate identical conformations of THP(A) and of THP(B) in H2O solution and in the crystal. THP(A) and THP(B) form zwitterionic molecules with the half-chair conformation. Both techniques, NMR and X-ray crystallography, indicate that the carboxyl group of THP(B) is in an axial position and that the carboxyl and the hydroxyl groups of THP(A) are also in axial positions. The coplanar zigzag configurations (H4 C4 C5 C6 H6) of THP(A) and THP(B), deduced from NMR coupling measurements, were confirmed by X-ray diffraction studies. The crystal structure of THP(B) indicates that the hydrogen atoms of the methyl group are disordered, exhibiting two distinct conformations, whereas the methyl group hydrogens of THP(A) exhibit only one conformation. This finding is in accordance with the dynamic properties of THP(A) and THP(B) derived from carbon spin-lattice relaxation rate measurements. In spite of a different crystal packing and considerable differences in the dynamic properties (T1) in solution, the two molecules exhibit an almost identical conformation. The similar conformation of the two different crystals can be considered as evidence for a relatively high internal stability of the molecule against intermolecular forces.

Hydrogen Bonding↗

PubMed1991

Formation of DNA triplexes accounts for arrests of DNA synthesis at d(TC)n and d(GA)n tracts.

To study the mechanism of arrest of DNA synthesis at d(TC)n and d(GA)n sequences, single-stranded DNA molecules including d(TC)27 or d(TC)31 tracts or a d(GA)27 tract were used as templates for in vitro assays of complementary DNA synthesis performed by extension of a primer with the Klenow polymerase or the Taq polymerase (Thermus aquaticus DNA polymerase). Electrophoresis of the products revealed that arrests occurred around the middle of these tracts. The arrests in the d(TC)n sequences were eliminated when dATP or dGTP was replaced with the analogue 7-deaza dATP or 7-deaza dGTP, respectively, or when the templates were preincubated with the Escherichia coli single-strand binding protein (SSB). Preincubation of the template including a d(GA)27 tract with SSB has also eliminated the arrests at this sequence. Furthermore, arrests did not occur at d[G(7-deaza A)]27 or d[(7-deaza G)A]27 tracts when molecules including such tracts were used as templates. These results are compatible with the notion that the arrests were caused by formation of d(TC)i.d(GA)i.d(TC)i and d(GA)i.d(GA)i.d(TC)i triplexes, in which the bases in the uncopied portions of the d(TC)n tracts, or of the d(GA)27 tract, and the purine bases in the newly synthesized d(TC)i.d(GA)i duplexes were bound by hydrogen bonds. In the assays performed with the Taq polymerase, the pH dependence (in the range of 6.0-9.0) and the temperature dependence of the arrests were determined. As the pH was lowered, the arrests in the d(TC)27 tract were enhanced, in line with the expected properties of d(TC)i.d(GA)i.d(TC)i triplexes. The arrests in the d(GA)27 tract were enhanced by an increase in the pH. At pH 7.2 the arrests in the d(GA)27 tract persisted up to 80 degrees C, whereas the arrests in the d(TC)27 tract were eliminated at 50 degrees C; these results presumably reflect the relative stabilities of the two triplexes mentioned above at this physiological pH value and could be biologically significant.

Adenine↗

PubMed1991

13C nuclear magnetic resonance and gas chromatography-mass spectrometry studies of carbon metabolism in the actinomycin D producer Streptomyces parvulus by use of 13C-labeled precursors.

Fructose and glutamate metabolism was monitored in cell suspensions of streptomyces parvulus by 13C nuclear magnetic resonance. The experiments were performed for cells grown with various 13C sources in a growth medium containing D-[U-13C]fructose, L-[13C]glutamate, or L-[U-13C]aspartate and with nonlabeled precursors to compare intracellular pools in S. parvulus cells at different periods of the cell life cycle. The transport of fructose into the cells was biphasic in nature; during rapid transport, mannitol, fructose, and glucose 6-phosphate were accumulated intracellularly, whereas during the passive diffusion of fructose, the intracellular carbohydrate pool comprised mainly trehalose (1,1'-alpha-alpha-D-glucose). The regulation of fructokinase activity by the intracellular intermediates may play an important role in fructose catabolism in S. parvulus. Transaldolase activity in S. parvulus was determined from the 13C nuclear magnetic resonance labeling pattern of trehalose carbons obtained from cells grown in medium containing either L-[U-13C]aspartate or L-[U-13C]glutamate. Only carbons 4, 5, and 6 of the disaccharide were labeled. Isotopomer analysis of the trehalose carbons led us to conclude that the flux through the reverse glycolytic pathway, condensation of glyceraldehyde 3-phosphate with dihydroxyacetone phosphate, makes at best a minor contribution to the 13C-labeled glucose units observed in trehalose. The pentose pathway and transaldolase activity can explain the labeling pattern of 4,5,6-13C3 of trehalose. Moreover, the transfer of the 13C label of L-[U-13C]aspartate into the different isotopomers of trehalose C4, C5, and C6 by the transaldolase activity allowed us to calculate the relative fluxes from oxaloacetate via gluconeogenesis and through the tricarboxylic acid cycle. The ratio of the two fluxes is approximately 1. However, the main carbon source for trehalose synthesis in S. parvulus is fructose and not glutamate or aspartate. The 13C enrichment and isotopomer population, measured by nuclear magnetic resonance and gas chromatography-mass spectrometry, of the actinomycin D peptide ring enabled us to specify the origins of the five amino acids of actinomycin D. Threonine and proline exhibited isotopomer populations similar to that of the extracellular L-[13C]glutamate, indicating that protein catabolism is the origin of their 13C label, whereas the isotopomer populations of sarcosine and N-methylvaline were similar to those of the new intracellular pool of S. parvulus that originated from D-[U-13C]fructose during the production of actinomycin D.

Carbohydrate Sequence↗

PubMed1990

Inherited disorders of carbohydrate metabolism in children studied by 13C-labelled precursors, NMR and GC-MS.

Glucose carbon recycling, glucose production and glucose turnover in glycogen storage disease type I and type II patients and control subjects were determined by a novel approach--mass isotopomer analysis of plasma 13C glucose. Changes in the isotopomer distribution of plasma 13C glucose were found only in glycogen storage disease type III patients and control subjects. Glucose carbon recycling parameters were also derived from 13C NMR spectra of plasma glucose C-1 splitting pattern. Our results eliminate a mechanism for glucose production in glycogen storage disease type I children involving gluconeogenesis. However, glucose release by amylo-1,6-glucosidase activity is in agreement with our results. A quantitative determination of the metabolic pathways of fructose conversion to glucose in normal children, and in children with disorders of fructose metabolism was derived from 13C NMR measurement of plasma 13C glucose isotopomer populations following [U-13C]fructose administration. A direct pathway from fructose, bypassing fructose-1-phosphate aldolase, to fructose-1,6-diphosphate in controls and hereditary fructose intolerant children (47% and 27%, respectively) was identified. In children with fructose-1,6-diphosphatase deficiency, only the gluconeogenic substrates were 13C labelled but no synthesis of glucose from [U-13C]fructose occurred. The significantly lower (by 68%) conversion of fructose to glucose in hereditary fructose intolerance, as compared to control subjects, and non-conversion in fructose-1,6-diphosphatase deficient subjects after [U-13C]fructose (approximately 20 mg/kg) administration can serve as the basis of a safe diagnostic test for patients suspected of inborn errors of fructose metabolism and other defects involving gluconeogenesis.

Blood Glucose↗

PubMed1990

Determination of fructose metabolic pathways in normal and fructose-intolerant children: a 13C NMR study using [U-13C]fructose.

An inborn deficiency in the ability of aldolase B to split fructose 1-phosphate is found in humans with hereditary fructose intolerance (HFI). A stable isotope procedure to elucidate the mechanism of conversion of fructose to glucose in normal children and in HFI children has been developed. A constant infusion of D-[U-13C]fructose was given nasogastrically to control and to HFI children. Hepatic fructose conversion to glucose was estimated by examination of 13C NMR spectra of plasma glucose. The conversion parameters in the control and HFI children were estimated on the basis of doublet/singlet values of the plasma beta-glucose C-1 splitting pattern as a function of the rate of fructose infusion (0.26-0.5 mg/kg per min). Significantly lower values (approximately 3-fold) for fructose conversion to glucose were obtained for the HFI patients as compared to the controls. A quantitative determination of the metabolic pathways of fructose conversion to glucose was derived from 13C NMR measurement of plasma [13C]glucose isotopomer populations. The finding of isotopomer populations of three adjacent 13C atoms at glucose C-4 (13C3-13C4-13C5) suggests that there is a direct pathway from fructose, by-passing fructose-1-phosphate aldolase, to fructose 1,6-bisphosphate. The metabolism of fructose by fructose-1-phosphate aldolase activity accounts for only approximately 50% of the total amount of hepatic fructose conversion to glucose. It is suggested that phosphorylation of fructose 1-phosphate to fructose 1,6-bisphosphate by 1-phosphofructokinase occurs in human liver (and intestine) when fructose is administered nasogastrically; 47% and 27% of the total fructose conversion to glucose in controls and in HFI children, respectively, takes place by way of this pathway. In view of the marked decline by 67% in synthesis of glucose from fructose in HFI subjects found in this study, the extent of [13C]glucose formation from a "trace" amount (approximately 20 mg/kg) of [U-13C]fructose infused into the patient can be used as a safe and noninvasive diagnostic test for inherent faulty fructose metabolism.

Blood Glucose↗

PubMed1989

(dT-dC)n and (dG-dA)n tracts arrest single stranded DNA replication in vitro.

Previous in vivo studies have indicated that (dT-dC)n.(dG-dA)n tracts (referred to here as (TC)n.(GA)n), which are widely dispersed in vertebrate genomes, may serve as pause or arrest signals for DNA replication and amplification. To determine whether these repeat elements act as stop signals for DNA replication in vitro, single stranded DNAs including (TC)n or (GA)n tracts of various lengths, were prepared by cloning such tracts into phage M13 vectors, and were replicated with the Klenow fragment of the E. coli DNA polymerase I, or with the calf thymus DNA polymerase alpha, by extension of an M13 primer. Gel electrophoresis of the reaction products revealed that the replication was specifically arrested around the middle of both (TC)n and (GA)n tracts of n greater than or equal to 16. However, whereas in the (TC)n tracts the arrests were less prominent at pH = 8.0 than at pH = 6.5-7.5, and were completely eliminated at pH = 8.5, the arrests in the (GA)n tracts were stronger at the higher pH values. These results, and previous data, suggest that the arrests were caused by formation of unusual DNA structures, possibly triple helices between partially replicated (TC)n or (GA)n tracts, and unreplicated portions of these sequences.

Animals↗

PubMed1989

Estimation of glucose carbon recycling in children with glycogen storage disease: A 13C NMR study using [U-13C]glucose.

A stable isotope procedure to estimate hepatic glucose carbon recycling and thereby elucidate the mechanism by which glucose is produced in patients lacking glucose 6-phosphatase is described. A total of 10 studies was performed in children with glycogen storage disease type I (GSD-I) and type III (GSD-III) and control subjects. A primed dose-constant nasogastric infusion of D-[U-13C]glucose (greater than 99% 13C-enriched) or an infusion diluted with nonlabeled glucose solution was administered following different periods of fasting. Hepatic glucose carbon recycling was estimated from 13C NMR spectra. The recycling parameters were derived from plasma beta-glucose C-1 splitting pattern, doublet/singlet values of plasma glucose C-1 in comparison to doublet/singlet values of known mixtures of [U-13C]glucose and unlabeled glucose as a function of 13C enrichment of glucose C-1. The fractional glucose C-1 enrichment of plasma glucose samples was analyzed by 1H NMR spectroscopy and confirmed by gas chromatography/mass spectroscopy. The values obtained for GSD-I patients coincided with the standard [U-13C]glucose dilution curve. These results indicate that the plasma glucose of GSD-I subjects comprises only a mixture of 99% 13C-enriched D-[U-13C]glucose and unlabeled glucose but lacks any recycled glucose. Significantly different glucose carbon recycling values were obtained for two GSD-III patients in comparison to GSD-I patients. Our results eliminate a mechanism for glucose production in GSD-I children involving gluconeogenesis. However, glucose release by amylo-1,6-glucosidase activity would result in endogenous glucose production of non-13C-labeled and nonrecycled glucose carbon, as was found in this study. In GSD-III patients gluconeogenesis is suggested as the major route for endogenous glucose synthesis. The contribution of the triose-phosphate pathway in these patients has been determined. The significant difference of the glucose C-1 splitting pattern in plasma GSD-III and control subjects, in comparison to GSD-I plasma, can be used as a parameter for estimating glucose recycling. This approach can be developed as a noninvasive diagnostic test for inborn enzymatic defects involving gluconeogenesis.

Carbon Isotopes↗

PubMed1989

Glucose recycling and production in glycogenosis type I and III: stable isotope technique study.

Glucose carbon recycling, endogenous glucose production, and glucose turnover rates were measured, by stable isotope methodology, in five patients with glycogen storage disease type I (GSD-I), two patients with glycogen storage disease type III (GSD-III), and three control children. A primed-constant infusion of D-[U-13C]glucose was administered nasogastrically to fasted subjects. The isotopic enrichments and 13C isotopomer distribution of plasma glucose were measured by chemical ionization gas chromatography mass spectroscopy. In response to increasing rates of glucose infusion, endogenous glucose production decreased, whereas the rate of glucose appearance or total glucose flux increased. Recycling of infused D-[U-13C]-glucose, calculated from changes in the isotopomer distribution of plasma [13C]glucose, was not detectable in GSD-I but reached 50% in GSD-III. In GSD-I the gluconeogenic pool was found to be highly labeled and recycled, whereas plasma glucose was diluted but not recycled. It is suggested that in GSD-I dilution of plasma glucose is due to release of glucose from branch points in glycogen. We propose that studies of the extent of glucose recycling and of isotopic enrichment of gluconeogenic precursors can be used as a noninvasive test for diagnosis of GSD-I and other defects in glucose production.

Blood Glucose↗

PubMed1988

The structure and biosynthesis of new tetrahydropyrimidine derivatives in actinomycin D producer Streptomyces parvulus. Use of 13C- and 15N-labeled L-glutamate and 13C and 15N NMR spectroscopy.

Two novel compounds, 2-methyl, 4-carboxy, 5-hydroxy-3,4,5,6-tetrahydropyrimidine (THP(A] and 2-methyl, 4-carboxy-3,4,5,6-tetrahydropyrimidine (THP(B] have been identified in the pool of Streptomyces parvulus by in vivo and in vitro studies. 13C and 15N were introduced into the compounds by feeding S. parvulus with 15N- and 13C-labeled L-glutamate. High resolution 13C and 15N NMR have been applied to elucidate their structure and biosynthesis in S. parvulus. The splitting patterns and coupling constants of adjacent nitrogen-carbon molecular fragments enable us to unravel their molecular structure. Two different glutamate pools are responsible for their biosynthesis, THP(A) carbon skeleton derives from the extracellular L-[13C]glutamate, whereas THP(B) stems from D-fructose via the intracellular glutamate. During cell growth, THP(A) is synthesized and becomes the major constituent of the intracellular pool. It is consumed after THP(B) is accumulated intracellularly. The onset of THP(A) and -(B) synthesis seems correlated to the time of actinomycin D synthesis. Their high cellular concentrations during actinomycin D synthesis suggest that they may function as nitrogen storage. Other possible functions of THP molecules within the cell are discussed.

Carbon Isotopes↗

PubMed1988

Microbial synthesis of L-[15N]leucine L-[15N]isoleucine, and L-[3-13C]-and L-[3'-13C]isoleucines studied by nuclear magnetic resonance and gas chromatography-mass spectrometry.

The preparation of leucine and isoleucine labeled with 15N and of site-specific 13C-labeled isoleucines is described. This method is based on the induction of the biosynthetic pathways specific for branched chain amino acids in glutamic acid producing bacteria, and controlled provision of stable isotope labeled precursors. Corynebacterium glutamicum (ATCC 13032), a glutamic acid overproducer, was incubated in leucine production medium which consisted of a basal medium supplemented with [15N]ammonium sulfate, glucose, and sodium alpha-ketoisocaproate. production of L-[15N]leucine reached 138 mumol/ml at an isotopic efficiency of 90%. It was purified and checked by proton NMR and GC-MS. The electron impact (EI) spectrum showed 95 atom% enrichment. The cultivation of C. glutamicum in a similar medium containing alpha-ketobutyrate yielded L-[15N]isoleucine at a concentration of 120 mumol/ml. The GC-MS EI and chemical ionization (CI) spectra confirmed enrichment of 96 atom% 15N as that of the labeled precursors. The biosynthesis of L-[13C]isoleucine was carried out by induced cells which were transferred to a similar medium in which [2-13C]- or [3-13C]pyruvic acid replaced glucose. 13C NMR of the product isoleucine revealed single-site enrichment at C-3 or at C-3' respective to the precursor [13C]pyruvate; i.e., C-3 was labeled from [2-13C]pyruvate and C-3' from [3-13C]pyruvate. Mass spectrometric analysis confirmed that all molecules were labeled only in one carbon. This site-specific incorporation of [13C]pyruvate is contrasted with the labeling pattern obtained when producing cells were supplied with [2-13C]acetate, instead of pyruvate, when most label was incorporated into carbons 3 and 3' of the same isoleucine molecule.

Actinomycetales↗

PubMed1988

Glucose recycling and production in children with glycogen storage disease type I, studied by gas chromatography/mass spectrometry and (U-13C)glucose.

Glucose recycling and production were determined in plasma of three children with glycogen storage disease type I (GSD-I) in comparison to normal children. A primed-constant infusion of (U-13C)glucose was introduced nasogastrically at different rates. Endogenous glucose production rates were found to be correlated with rates of glucose infusion. A range of glucose production of 3.5-1.8 mg kg-1 min-1 was found when glucose infusion rates increased from 0.13 to 6 mg kg-1 min-1. The isotopomer distribution of the infused (U-13C)glucose and the plasma glucose in GSD-I children, measured by chemical ionization gas chromatography/mass spectrometry, were identical, indicating absence of glucose recycling. However, a significant change in the isotopomer distribution of plasma glucose was observed in normal subjects. It is suggested that the origin of endogenous glucose production in GSD-I children, during (U-13C)glucose infusion, is from non-labelled sources. The absence of glucose recycling is indicative of a deficiency of glucose 6-phosphatase activity in the liver of GSD-I patients.

Alanine↗

PubMed1988

Metabolic regulation in Streptomyces parvulus during actinomycin D synthesis, studied with 13C- and 15N-labeled precursors by 13C and 15N nuclear magnetic resonance spectroscopy and by gas chromatography-mass spectrometry.

Recent studies have suggested that the onset of synthesis of actinomycin D in Streptomyces parvulus is due to a release from L-glutamate catabolic repression. In the present investigation we showed that S. parvulus has the capacity to maintain high levels of intracellular glutamate during the synthesis of actinomycin D. The results seem contradictory, since actinomycin D synthesis cannot start before a release from L-glutamate catabolic repression, but a relatively high intracellular pool of glutamate is needed for the synthesis of actinomycin D. Utilizing different labeled precursors, D-[U-13C]fructose and 13C- and 15N-labeled L-glutamate, and nuclear magnetic resonance techniques, we showed that carbon atoms of an intracellular glutamate pool of S. parvulus were not derived biosynthetically from the culture medium glutamate source but rather from D-fructose catabolism. A new intracellular pyrimidine derivative whose nitrogen and carbon skeletons were derived from exogenous L-glutamate was obtained as the main glutamate metabolite. Another new pyrimidine derivative that had a significantly reduced intracellular mobility and that was derived from D-fructose catabolism was identified in the cell extracts of S. parvulus during actinomycin D synthesis. These pyrimidine derivatives may serve as a nitrogen store for actinomycin D synthesis. In the present study, the N-trimethyl group of a choline derivative was observed by 13C nuclear magnetic resonance spectroscopy in growing S. parvulus cells. The choline group, as well as the N-methyl groups of sarcosine, N-methyl-valine, and the methyl groups of an actinomycin D chromophore, arose from D-fructose catabolism. The 13C enrichments found in the peptide moieties of actinomycin D were in accordance with a mechanism of actinomycin D synthesis from L-glutamate and D-fructose.

Chemical Phenomena↗

PubMed1987

A quantitative analysis of the metabolic pathways of hepatic glucose synthesis in vivo with 13C-labeled substrates.

A quantitative analysis of the major metabolic pathways of hepatic glucose synthesis in fasted rats was conducted. [2-13C]Acetate was administered intraintestinally into awake fasted rats. 13C NMR and GC-MS analysis were used to quantitate the isotopic enrichments of glutamate, glutamine, lactate, alanine and the newly synthesized liver glucose. By measuring the ratio of carbon atoms in glutamate molecules derived from acetyl-CoA to carbon atoms in the glucose molecule derived from oxaloacetate and gluconeogenic substrates, such as lactate and alanine, the relative activities of the Krebs cycle and gluconeogenesis were quantified. Our results indicate that the percentage of glucose carbons originating by 'metabolic exchange' with the oxaloacetate pool, via the Krebs cycle, is less than 7%.

Acetates↗

PubMed1987

13C-NMR, 1H-NMR and gas-chromatography mass-spectrometry studies of the biosynthesis of 13C-enriched L-lysine by Brevibacterium flavum.

The basic metabolic pathways of lysine biosynthesis in Brevibacterium flavum, a strain which excretes excessive amounts of L-lysine, have been followed by using two 13C-labeled precursors. 13C- and 1H-NMR spectroscopies in conjunction with gas chromatography mass spectrometry (GC-MS) have revealed the various metabolic pathways leading to L-[13C]lysine. Discrete metabolic pathways give rise to distinct labeling patterns. L-Lysine resulting from [1-13C]glucose fermentation is relatively specifically labeled: L-[3,5-13C]lysine is the main product. Experimental and theoretical approaches based on the 13C-enrichment values of intracellular glutamate, a major intermediate metabolite, allowed us to assess the relative contribution of the major metabolic pathways forming lysine. The labeling pattern of glutamate reflects the isotope distribution in 2-oxoglutarate. When [2-13C]acetate is used as the sole carbon source in the culture, the energy-producing steps of the Krebs cycle are essential. The higher activity of the Krebs cycle, when endogenous carbohydrates are exhausted from the culture, is indicated by the increased 13C enrichment in C-1 of lysine and reveal a high content of isotopomers of four, five and six 13C atoms in the lysine molecule, pointing out that the four-carbon intermediates of the cycle are being derived from the glyoxylate shunt pathway. Such a phenomenon does not occur in glucose fermentation. GC-MS analyses of 13C enrichments and isotopomer distributions in metabolites and end products are in good agreement with the predicted contribution of each metabolic pathway. This new methodological approach of combined NMR and GC-MS has been demonstrated to be applicable to various other metabolic studies.

Brevibacterium↗

PubMed1987

'Illegitimate' recombination events in polyoma-transformed rat cells.

In the LPT line of polyoma (Py)-transformed rat cells, amplification of the integrated viral DNA and of cell nucleotide sequences flanking the viral integration site, can be induced either spontaneously or by treatment with carcinogens. We show here that the amplified DNA includes interspersed viral and cellular sequences generated by 'illegitimate' recombination events. Genomic libraries have been prepared in phage lambda vectors from LPT cells treated with the inducing agent mitomycin C and from untreated LPT cells. Four phages, including viral-cell DNA recombinants, have been isolated from these libraries. Sequencing through the recombination sites revealed the following characteristics: (i) The crossover points map at four different positions in the viral DNA and at four different positions in the flanking cell DNA. (ii) There are very short homologous sequences of 1, 2, or 4 bp, at the recombination sites. (iii) Aside from the exchanges between the viral and the cellular DNA, no further rearrangements occurred around the new viral-cellular DNA junctions. (iv) Next to the recombination sites, there are blocks of homopurine-homopyrimidine sequences, which may assume a structure that differs from the Watson-Crick double helix. (v) Clustered homologous sequence blocks of up to 10 bp are present less than 200 bp away from the recombination sites. These homologies are not in register. Based on these results, we propose a model that may account for these recombination events and, more generally, for recombination events that occur during gene amplification in mammalian cells.

Animals↗

PubMed1987

Unusual sequence element found at the end of an amplicon.

In a polyomavirus-transformed rat cell line, designated LPT, the polyomavirus DNA is integrated into a single chromosomal site. Treatment of LPT cells with carcinogens induces amplification of the integrated virus DNA and flanking cellular sequences. We show that the amplification is arrested within a specific cell DNA segment that maps 1.3 to 1.85 kilobases beyond one virus-cell DNA junction, defined as the left junction. We also present the sequence of an 897-base-pair fragment spanning the arrest site. This fragment contains an unusual sequence element, which consists of two contiguous components, a potential cruciform with stems of 6 base pairs and a d(G-A)27 X d(T-C)27 tract, and maps 1,497 to 1,564 nucleotides beyond the left junction. The possibility that this unusual sequence plays a role in the arrest of the amplification process is discussed.

Animals↗