A new antitumor antibiotic, FR900840. I. Discovery, identification, isolation and characterization.

A new antitumor antibiotic, FR900840, was isolated from a culture broth of Streptomyces strain No. 8727 as a pale yellowish prism and the molecular formula was determined to be C7H11N3O5. The antibiotic exhibited prominent antitumor effects on human tumor cell lines both in vitro and in vivo.

Synergistic inhibition of hepatic ketogenesis in the presence of insulin and a cAMP antagonist.

The separate or combined effects of insulin and the cAMP antagonist, the Rp-diastereomer of adenosine cyclic 3',5'-phosphorothioate (Rp-cAMPS), were examined on fatty acid-stimulated ketogenesis in hepatocytes from normal fasted rats. Addition of 0.4 mM oleic acid or 0.4 mM octanoic acid resulted in a linear increase in ketone production measured over 60 min. When oleic acid was the substrate, incubation with 1 to 30 microns Rp-cAMPS alone or 0.1 to 10 nM insulin alone caused a variable decrease in the production of ketones which did not exceed an average value of 30% in any one experiment. The simultaneous addition of Rp-cAMPS and insulin resulted in a greater than additive inhibition which reached average values between 47-60% when the theoretical combined inhibitory effect of the insulin alone plus the Rp-cAMPS alone was less than 18%. No significant effects of either insulin or Rp-cAMPS, alone or in combination, were seen when octanoic acid was the substrate. These data imply that Rp-cAMPS can potentiate insulin inhibition of hepatic ketogenesis through inhibition of a cAMP-mediated process.

Pseudoketogenesis in the perfused rat heart.

Ketogenesis is usually measured in vivo by dilution of tracers of (3R)-hydroxybutyrate or acetoacetate. We show that, in perfused working rat hearts, the specific activities of (3R)-hydroxybutyrate and acetoacetate are diluted by isotopic exchanges in the absence of net ketogenesis. We call this process pseudoketogenesis. When hearts are perfused with buffer containing 2.3 mM of [4-3H]- plus [3-14C]acetoacetate, the specific activities of [4-3H] and [3-14C]acetoacetate decrease while C-1 of acetoacetate becomes progressively labeled with 14C. This is explained by the reversibility of reactions catalyzed by mitochondrial 3-oxoacid-CoA transferase and acetoacetyl-CoA thiolase. After activation of labeled acetoacetate, the specific activity of acetoacetyl-CoA is diluted by unlabeled acetoacetyl-CoA derived from endogenous fatty acids or glucose. Acetoacetyl-CoA thiolase partially exchanges 14C between C-1 and C-3 of acetoacetyl-CoA. Finally, 3-oxoacid-CoA transferase liberates weakly labeled acetoacetate which dilutes the specific activity of extracellular acetoacetate. An isotopic exchange in the reverse direction is observed when hearts are perfused with unlabeled acetoacetate plus [1-14C]-, [13-14C]-, or [15-14C]palmitate; here also, acetoacetate becomes labeled on C-1 and C-3. Computations of specific activities of (3R)-hydroxybutyrate, acetoacetate, and acetyl-CoA yield minimal rates of pseudoketogenesis ranging from 19 to 32% of the net uptake of (3R)-hydroxybutyrate plus acetoacetate by the heart.

Cloning of the Alcaligenes eutrophus genes for synthesis of poly-beta-hydroxybutyric acid (PHB) and synthesis of PHB in Escherichia coli.

Eight mutants of Alcaligenes eutrophus defective in the intracellular accumulation of poly-beta-hydroxybutyric acid (PHB) were isolated after transposon Tn5 mutagenesis with the suicide vector pSUP5011. EcoRI fragments which harbor Tn5-mob were isolated from pHC79 cosmid gene banks. One of them, PPT1, was used as a probe to detect the intact 12.5-kilobase-pair EcoRI fragment PP1 in a lambda L47 gene bank of A. eutrophus genomic DNA. In six of these mutants (PSI, API, GPI, GPIV, GPV, and GPVI) the insertion of Tn5-mob was physically mapped within a region of approximately 1.2 kilobase pairs in PP1; in mutant API, cointegration of vector DNA has occurred. In two other mutants (GPII and GPIII), most probably only the insertion element had inserted into PP1. All PHB-negative mutants were completely impaired in the formation of active PHB synthase, which was measured by a radiometric assay. In addition, activities of beta-ketothiolase and of NADPH-dependent acetoacetyl coenzyme A (acetoacetyl-CoA) reductase were diminished, whereas the activity of NADPH-dependent acetoacetyl-CoA reductase was unaffected. In all PHB-negative mutants the ability to accumulate PHB was restored upon complementation in trans with PP1. The PHB-synthetic pathway of A. eutrophus was heterologously expressed in Escherichia coli. Recombinant strains of E. coli JM83 and K-12, which harbor pUC9-1::PP1, pSUP202::PP1, or pVK101::PP1, accumulated PHB up to 30% of the cellular dry weight. Crude extracts of these cells had significant activities of the enzymes PHB synthase, beta-ketothiolase, and NADPH-dependent acetoacetyl-CoA reductase. Therefore, PP1 most probably encodes all three genes of the PHB-synthetic pathway in A. eutrophus. In addition to PHB-negative mutants, we isolated mutants which accumulate PHB at a much lower rate than the wild type does. These PHB-leaky mutants exhibited activities of all three PHB-synthetic enzymes; Tn5-mob had not inserted into PP1, and the phenotype of the wild type could not be restored with fragment PP1. The rationale for this mutant type remains unknown.

Cloning and expression in Escherichia coli of the Alcaligenes eutrophus H16 poly-beta-hydroxybutyrate biosynthetic pathway.

The poly-beta-hydroxybutyrate (PHB) biosynthetic pathway from Alcaligenes eutrophus H16 has been cloned and expressed in Escherichia coli. Initially, an A. eutrophus H16 genomic library was constructed by using cosmid pVK102, and cosmid clones that encoded the PHB biosynthetic pathway were sought by assaying for the first enzyme of the pathway, beta-ketothiolase. Six enzyme-positive clones were identified. Three of these clones manifested acetoacetyl coenzyme A reductase activity, the second enzyme of the biosynthetic pathway, and accumulated PHB. PHB was produced in the cosmid clones at approximately 50% of the level found in A. eutrophus. One cosmid clone was subjected to subcloning experiments, and the PHB biosynthetic pathway was isolated on a 5.2-kilobase KpnI-EcoRI fragment. This fragment, when cloned into small multicopy vectors, can direct the synthesis of PHB in E. coli to levels approaching 80% of the bacterial cell dry weight.

Ketopantoic acid reductase of Pseudomonas maltophilia 845. Purification, characterization, and role in pantothenate biosynthesis.

Ketopantoic acid reductase (EC 1.1.1.169), an enzyme that catalyzes the formation of D-(-)-pantoic acid from ketopantoic acid, was purified 6,000-fold to apparent homogeneity with a 35% overall recovery from Pseudomonas maltophilia 845 and then crystallized. The relative molecular mass of the native enzyme, as estimated by the sedimentation equilibrium method, is 87,000 +/- 5,000, and the subunit molecular mass is 30,500. The enzyme shows high specificity for ketopantoic acid as a substrate (Km = 400 microM, Vm = 1,310 units/mg of protein) and NADPH as a coenzyme (Km = 31.8 microM). Only 2-keto-3-hydroxyisovalerate (Km = 8.55 mM, Vm = 35.8 units/mg) was reduced among a variety of other carbonyl compounds tested. The reaction is reversible (Km for D-(-)-pantoic acid = 52.1 mM), although the reaction equilibrium greatly favors the direction of D-(-)-pantoic acid formation. That the enzyme is responsible for the synthesis of D-(-)-pantoic acid necessary for the biosynthesis of pantothenic acid in P. maltophilia 845 is indicated by the observations that only this enzyme is missing in D-(-)-pantoate (or pantothenate)-requiring mutants derived from P. maltophilia 845 among several enzymes (i.e. ketopantoyl lactone reductase (EC 1.1.1.168) and acetohydroxy acid isomeroreductase (EC 1.1.1.86], which may be concerned in the formation of D-(-)-pantoic acid, assayed, whereas it is present in substantial amounts in the parent strain and in spontaneous revertants of the mutants.

Purification and characterization of NADP-linked acetoacetyl-CoA reductase from Zoogloea ramigera I-16-M.

An NADP-linked acetoacetyl-CoA reductase was purified to electrophoretic homogeneity from Zoogloea ramigera I-16-M, a poly(3-hydroxybutyrate)-accumulating bacterium. The purified enzyme showed specific activity of 412 mumol acetoacetyl-CoA reduced per min per mg protein, which constituted an 880-fold purification compared to the crude extract, with a 32% yield. Electrophoretic analysis of the purified enzyme which had been cross-linked with dimethylsuberimidate showed that the native enzyme (Mr 92,000) is a tetramer of four identical subunits (Mr 25,500). Among the various D-(-)- and L-(+)-3-hydroxyacyl-CoAs tested, the purified enzyme oxidized only D-(-)-3-hydroxybutyryl-CoA and to a lesser extent D-(-)-3-hydroxyvaleryl-CoA in the presence of NADP+. The antiserum prepared against the purified enzyme completely inhibited poly(3-hydroxybutyrate) synthesis from acetyl-CoA by a crude extract of Z. ramigera I-16-M cells. These findings indicate that this enzyme plays an indispensable role as the supplier of D-(-)-3-hydroxybutyryl-CoA in poly(3-hydroxybutyrate) synthesis in this bacterium.

A method for simultaneous determination of the two possible products of acetohydroxy acid synthase.

A method for the simultaneous assay of 2-acetolactate and 2-aceto-2-hydroxybutyrate formation catalyzed by acetohydroxy acid synthase in the presence of its substrates pyruvate and 2-ketobutyrate is described. The method, appropriate for the study of the physiologically and mechanistically significant competition between the two reactions, involves oxidative decarboxylation of the acetohydroxy acids to the corresponding 2,3-diketones, transfer of the volatile diketones to methanol, and gas chromatographic analysis with electron-capture detection. Oxidative decarboxylation by air requires catalytic activation, and addition of iron salts is crucial to the success of the method with purified enzymes.

Poly-beta-hydroxybutyrate membrane structure and its relationship to genetic transformability in Escherichia coli.

The effects of competence-inducing treatments on the composition and organization of membrane lipids in Escherichia coli K-12, DH1, DH5, HB101, and RR1 were investigated for two widely used protocols in which transformability is developed at low temperatures in Ca2+ buffers. At stages during each procedure, the lipid compositions of the cells were determined, and the thermotropic lipid phase transitions were observed in whole cell culture by fluorescence assay with the hydrophobic probe N-phenyl-1-naphthylamine. Competence was evaluated by determining transformation efficiencies with plasmid pBR322 DNA. The competence-inducing procedures effected only slight changes in phospholipid compositions which did not correlate with transformability. However, the induction of competence was coincident with de novo synthesis and incorporation of poly-beta-hydroxybutyrate into the cytoplasmic membranes and with the appearance of a sharp lipid phase transition above physiological temperatures. Transformation efficiencies correlated with poly-beta-hydroxybutyrate concentrations and with the intensity of the new phase transition. Transformability, poly-beta-hydroxybutyrate synthesis and the new phase transition were not significantly affected by inhibition of protein synthesis with chloramphenicol or inhibition of respiration or ATP synthesis with azide, cyanide, arsenate, or 2,4-dinitrophenol; however, when poly-beta-hydroxybutyrate synthesis was inhibited with acetaldehyde, the new phase transition was not observed, and competence failed to develop. These studies suggest that genetic transformability in E. coli may be physiologically regulated.

Role of small subunit (IlvN polypeptide) of acetohydroxyacid synthase I from Escherichia coli K-12 in sensitivity of the enzyme to valine inhibition.

Most of the coding sequence for the IlvN polypeptide subunit of acetohydroxyacid synthase I was deleted from the ilvB+ ilvN+ plasmid pTCN12 by in vitro methods. Several ilvB+ delta ilvN derivatives of pTCN12 were identified among transformants of a strain otherwise lacking any acetohydroxyacid synthase. Deletion derivatives produced an enzymatically active IlvB polypeptide, as shown by the Ilv+ phenotype of transformed cells and by immunologic and enzymatic assays. However, whereas the growth of pTCN12 transformants was sensitive to valine inhibition, growth of the ilvB+ delta ilvN transformants was relatively resistant. Moreover, in vitro analyses confirmed that both acetolactate and acetohydroxybutyrate synthesis in extracts of the ilvB+ delta ilvN transformants was resistant to valine inhibition, in comparison with that in extracts of pTCN12 transformants or with that catalyzed by purified acetohydroxyacid synthase I. The IlvN polypeptide had a minimal effect, if any, on IlvB polypeptide accumulation as measured by immunoprecipitation, but its absence resulted in a greater than 10-fold reduction in enzyme specific activity.

Acetoacetate and glucose as lipid precursors and energy substrates in primary cultures of astrocytes and neurons from mouse cerebral cortex.

Primary cultures of astrocytes and neurons derived from neonatal and embryonic mouse cerebral cortex, respectively, were incubated with [3-14C]acetoacetate or [2-14C]glucose. The utilization of glucose and acetoacetate, the production of lactate, D-3-hydroxybutyrate, and 14CO2, and the incorporation of 14C and of 3H from 3H2O into lipids and lipid fractions were measured. Both cell types used acetoacetate as an energy substrate and as a lipid precursor; lactate was the major product of glucose metabolism. About 60% of the acetoacetate that was utilized by neurons was oxidized to CO2, whereas this was only approximately 20% in the case of cultured astrocytes. This indicates that the rate at which 14C-labeled Krebs cycle intermediates exchange with pools of unlabeled intermediates is much higher in astrocytes than in neurons. Acetoacetate is a better precursor for the synthesis of fatty acids and cholesterol than glucose, presumably because it can be used directly in the cytosol for these processes; preferential incorporation into cholesterol was not observed in these in vitro systems. We conclude that ketone bodies can be metabolized both by the glial cells and by the neuronal cells of developing mouse brain.

Evidence for impaired metabolism in liver during induced lactation ketosis of dairy cows.

Ketosis was induced in four lactating dairy cows at an average of 36 d postpartum and 24 d after initiation of a protocol for inducing ketosis. Liver and adipose biopsies were taken at five stages; once prepartal, once early postpartal, once during an induction protocol, once during ketosis, and once during recovery after treatment of ketosis. At each stage, in vitro estimates were made of capacities of liver slices for gluconeogenesis, fatty acid oxidation, and ketogenesis and of capacities of adipose tissue slices for lipogenesis and lipolysis. There were no significant differences for any metabolic capacity between prepartal and early postpartal stages; however, a significant decrease in hepatic metabolic activity was associated with ketosis. Gluconeogenic rates from all substrates tested were decreased significantly during ketosis, and similar decreases were measured for oxidation of gluconeogenic substrates and fatty acids. Treatment of ketosis resulted in complete recovery from impairments of metabolism in liver. Results suggest that a prolonged energy deficit, plus a major influx of ketone precursors, is accompanied by eventual hepatic impairment, that clinical ketosis is associated with this impairment, and that the impairment is reversed by effective treatment of ketosis.

The effect of the dissolved oxygen concentration and anabolic limitations on the behaviour of Rhizobium ORS571 in chemostat cultures.

Chemostat cultures of Rhizobium ORS571 limited by the supply of oxygen or an anabolic substrate contained poly-beta-hydroxybutyrate (PHB). Low amounts of PHB (about 10%) were present in ammonia- or nitrate-limited cultures; higher amounts were found in Mg++-limited cultures (about 20%) and in oxygen-limited nitrogen-fixing cultures (37%). A method is described to calculate YATP values (g PHB-free biomass . mol-1 ATP) from the Ysucc values (g dry wt . mol-1 succinate) measured. Ysucc and YATP values in cultures limited by the supply of an anabolic substrate and in the oxygen-limited ammonia-assimilating culture were much lower than the values found in the PHB-free succinate-limited cultures. This shows that uncoupling of growth and energy production occurred. Therefore, H2/N2 ratio (mol hydrogen formed per mol nitrogen fixed) in nitrogen-fixing cultures could not be calculated from the comparison of the YATP value found in the nitrogen-fixing culture and the value found in the corresponding ammonia-assimilating culture. Although the optimal dissolved oxygen concentration (d.o.c.) for nitrogen-fixing cultures of Rhizobium ORS571 is 5 or 10 microM, nitrogen-fixing cultures could be obtained up to a d.o.c. of 40 microM. Not only nitrogenase but also hydrogenase was active at this d.o.c. However, accumulation of PHB (10%) may indicate that cultures grown at unfavourable oxygen concentrations (15-40 microM O2) were N-limited rather than energy-limited, which may be the result of partial inactivation or repression of nitrogenase at a higher d.o.c.

Differential effects of alanine on ketogenesis and triacylglycerol formation by isolated perfused livers from euthyroid and hyperthyroid rats.

We examined the effects of infusion of alanine on hepatic concentration of glycero-3-phosphate (glycero-3-P) and output of triacylglycerol (TG) by isolated perfused livers from triiodothyronine (T3)-treated rats. It was expected that because of its gluconeogenic and antiketogenic properties, alanine might stimulate accumulation of glycero-3-P, which in turn might result in enhanced TG production by the hyperthyroid livers. The hepatic concentration of glycero-3-P is lower in livers from T3-treated rats than in euthyroid rats. Infusion of 1.83 and 4.23 mmol alanine/4 h did not alter the hepatic concentration of glycero-3-P and output of triacylglycerol by livers from T3-treated rats. However in these livers, the two concentrations of infused alanine increased the output of glucose and decreased the output of ketone bodies. In livers from euthyroid animals, the infusion of 1.83 mmol alanine/4 h had no effect, whereas 4.23 mmol/4 h decreased hepatic concentration of glycero-3-P. These concentrations of alanine did not alter the output of ketone bodies or TG, but progressively decreased the output of glucose by the euthyroid livers. Our data suggested that the decreased availability of glycero-3-P in livers from T3-treated rats is rate-limiting for TG production and correlated with the diminished output of TG, whereas in livers from euthyroid rats, the glycero-3-P concentration is sufficient to maintain maximal synthesis and secretion of TG. Furthermore, the effects of alanine on the output of ketone bodies or glucose appear to be independent of its effects on hepatic glycero-3-P.

Ketogenic regulation by certain metabolites in rumen epithelium.

In experiments with rumen epithelium incubated in vitro in the presence of butyrate, the ketogenic effect of glucose was shared by epimeric monosaccharides but not by non-metabolizable analogues. 14C from glucose was not incorporated into ketone bodies. Malate increased ketogenesis from butyrate and decreased its oxidation, pyruvate and NH4+ had the opposite effect, and malonate inhibited both processes. The ketogenic effect of glucose was also effective with isovalerate maintaining the high proportion of acetoacetate which is characteristic of this substrate. Rumen epithelium transformed added acetoacetate into 3-hydroxybutyrate. It is concluded that reducing equivalents produced from glucose and other metabolizable substrates are responsible regulators of ketogenesis from butyrate. The results are discussed in view of the functional role of ruminal ketogenesis.

Ketogenesis in the living rat followed by 13C NMR spectroscopy.

The metabolic fate of 13C1-labeled butyrate in the liver of living rats has been studied by 13C NMR. The formation of the ketone bodies acetoacetate and beta-hydroxybutyrate was observed in vivo as well as resonances from glutamate, glutamine, and carbonate. The observed time course of these metabolites demonstrates the potential of the technique to measure enzyme kinetics in vivo and also to measure the enzyme capacity of a given organ to metabolize a substrate. The in vivo spectra were compared to in vitro spectra of the excised liver and perchloric acid extracts of the liver. Observation of the metabolites and monitoring of their time course in vivo would not have been possible without distinct improvements in the spectral resolution and the spatial localization of the radio-frequency field within the liver. As a novel approach, we have selected the carbonyl region of the 13C NMR spectra for identifying the ketone bodies and other oxidation products in the liver.

Role of acetohydroxy acid isomeroreductase in biosynthesis of pantothenic acid in Salmonella typhimurium.

Structural genes have been identified for all of the enzymes involved in the biosynthesis of pantothenic acid in Salmonella typhimurium and Escherichia coli K-12, with the exception of ketopantoic acid reductase, which catalyzes the conversion of alpha-ketopantoate to pantoate. The acetohydroxy acid isomeroreductase from S. typhimurium efficiently bound alpha-ketopantoate (K(m) = 0.25 mM) and catalyzed its reduction at 1/20 the rate at which alpha-acetolactate was reduced. Since two enzymes could apparently participate in the synthesis of pantoate, a S. typhimurium ilvC8 strain was mutagenized to derive strains completely blocked in the conversion of alpha-ketopantoate to pantoate. Several isolates were obtained that grew in isoleucine-valine medium supplemented with either pantoate or pantothenate, but not in the same medium supplemented with alpha-ketopantoate or beta-alanine. The mutations that conferred pantoate auxotrophy (designated panE) to these isolates appeared to be clustered, but were not linked to panB or panC. All panE strains tested had greatly reduced levels of ketopantoic acid reductase (3 to 12% of the activity present in DU201). The capacity of the isomeroreductase to synthesize pantoate in vivo was assessed by determining the growth requirements of ilvC(+) derivatives of panE ilvC8 strains. These strains required either alpha-ketopantoate, pantoate, or pantothenate when the isomeroreductase was present at low levels; when the synthesis of isomeroreductase was induced, panE ilvC(+) strains grew in unsupplemented medium. These phenotypes indicate that a high level of isomeroreductase is sufficient for the synthesis of pantoate. panE ilvC(+) strains also grew in medium supplemented with lysine and methionine. This phenotype resembles that of some S. typhimurium ilvG mutants (e.g., DU501) which are partially blocked in the biosynthesis of coenzyme A and are limited for succinyl coenzyme A. panE ilvC(+) strains which lack the acetohydroxy acid synthases required only methionine for growth (in the presence of leucine, isoleucine, and valine). This and other evidence suggested that the synthesis of pantoic acid by isomeroreductase was blocked by the alpha-acetohydroxy acids and that pantoic acid synthesis was enhanced in the absence of these intermediates, even when the isomeroreductase was at low levels. panE ilvC(+) strains reverted to pantothenate independence. Several of these revertants were shown to have elevated isomeroreductase levels under noninduced and induced conditions; the suppressing mutation in each revertant was shown to be closely linked to ilvC by P22 transduction. This procedure presents a means for obtaining mutants with altered regulation of isomeroreductase.

Studies on the relation of gamma-hydroxybutyric acid (GHB) to gamma-aminobutyric acid (GABA). Evidence that GABA is not the sole source for GHB in rat brain.

The effects of gamma-aminobutyric acid (GABA)-alpha-oxoglutarate aminotransferase (GABA-T) inhibitors, L-glutamic acid decarboxylase (GAD) inhibitors, and antipetit mal anticonvulsants on gamma-hydroxybutyric acid (GHB) and GABA were studied. Treatment with anticonvulsants and GABA-T inhibitors resulted in an increase in steady-state brain levels of both GHB and GABA. GAD inhibitors produced markedly decreased levels of brain GABA but no change in GHB concentrations. Studies of GHB derived exclusively from GABA showed that GABA-T inhibitors which produced an elevation of steady-state levels of GHB in brain also resulted in a decrease in GABA-derived GHB. Intracerebroventricular (i.c.v.) administration of GABA, putrescine, and 1,4-butanediol all produced significant elevations in brain GHB, but GABA-T inhibitors blocked this effect of GABA and putrescine. These data suggest that there may be another source for GHB in brain in addition to GABA and raise the possibility that 1,4-butanediol may be that source.