Addition Of Succinate Research Articles

Enhancement, by succinate addition, of the production of cloned glucoamylase from recombinant yeast using a SUC2 promoter

The effect of succinate on the production of cloned glucoamylase by a recombinant yeast system was investigated. Saccharomyces cerevisiae was transformed with the 2 μ-based plasmid YEpSUCSTA containing the SUC2 promoter, the STA signal sequence, and the STA structural gene. Specific glucoamylase expression was 89% higher in a semi-synthetic medium containing succinate than in medium without succinate. This increase in glucoamylase activity in succinate-containing media reflects two roles of succinate; protection of rapid decrease in pH in the culture broth and enhancement of glucoamylase biosynthesis.

Genetic Analysis of Mutations Affecting pckA Regulation in Rhizobium (Sinorhizobium) meliloti

The enzyme phosphoenolpyruvate carboxykinase (Pck) catalyzes the first step in the gluconeogenic pathway in most organisms. We are examining the genetic regulation of the gene encoding Pck, pckA, in Rhizobium (now Sinorhizobium) meliloti. This bacterium forms N2-fixing root nodules on alfalfa, and the major energy sources supplied to the bacteria within these nodules are C4-dicarboxylic acids such as malate and succinate. R. meliloti cells growing in glucose minimal medium show very low pckA expression whereas addition of succinate to this medium results in a rapid induction of pckA transcription. We identified spontaneous mutations (rpk) that alter the regulation of pckA expression such that pckA is expressed in media containing the non-inducing carbon sources lactose and glucose. Genetic and phenotypic analysis allowed us to differentiate at least four rpk mutant classes that map to different locations on the R. meliloti chromosome. The wild-type locus corresponding to one of these rpk loci was cloned by complementation, and two Tn5 insertions within the insert DNA that no longer complemented the rpk mutation were identified. The nucleotide sequence of this region revealed that both Tn5 insertions lay within a gene encoding a protein homologous to the GalR/LacI family of transcriptional regulators that are involved in metabolism.

Modification of swelling-contraction-aggregation processes in rat muscle mitochondria by the 1,4-dihydropyridines, cerebrocrast and glutapyrone, themselves and in the presence of azidothymidine.

The influence of the 1,4-dihydropyridines (DHPs), water-soluble glutapyrone available as sodium, potassium and ammonium salts of 2-(2,6-dimethyl-3,5-diethoxycarbonyl-1,4-DHP-4-carboxamide)glutaric acid, from one side, and a lipophylic cerebrocrast, 2-propoxyethyl 2,6-dimethyl-4-(2-difluoromethoxyphenyl)-1,4-DHP-3,5-dicarboxylate, from the other side, on partially damaged mitochondria of the Wistar rat hindlimb muscle was also studied. The following tests were made: (1) rates of endogenous respiration and substrate (succinate) oxidation and oxidative phosphorylation; (2) rates and amplitudes of high-amplitude swelling and contraction after the addition of ATP, ADP and succinate to the previously swollen mitochondria and (3) rate of reversible self-aggregation of mitochondria isolated in salt media after ATP-induced contraction without and in the presence of azidothymidine (AZT). Cerebrocrast (10-100 microM) partially normalized the endogenous respiration rate and slightly augmented the respiration rate after the addition of succinate and to lesser extent ADP. Cerebrocrast in a concentration-dependent manner (2.5-50 microM) increased (two-fold at 20-50 microM) the active contraction amplitude of swollen mitochondria, induced by single or repeated additions of ATP. The influence of cerebrocrast on the ADP- and succinate-induced contractions was less obvious. Unlike cerebrocrast glutapyrone caused a reduction of the ATP-induced contraction amplitude (two-fold at 0.5-5.0 mM), not impairing the mitochondrial contraction ability in response to ATP or succinate. Pre-exposure to 2.5 mM glutapyrone resulted in at least a 10-fold inhibition of the reversible aggregation rate in the presence of 99 and 198 microM AZT. The results suggest the usefulness of further study of cerebrocrast and glutapyrone in preventing AZT-induced and some other mitochondrial myopathies.

Osmoprotectant-dependent expression of plcH, encoding the hemolytic phospholipase C, is subject to novel catabolite repression control in Pseudomonas aeruginosa PAO1.

Expression of the hemolytic phospholipase C (PlcH) of Pseudomonas aeruginosa is induced under phosphate starvation conditions or in the presence of the osmoprotectants choline and glycine betaine. Because choline and glycine betaine may serve as carbon and energy sources in addition to conferring osmoprotection to P. aeruginosa, it seemed possible that induction of plcH is subject to catabolite repression control (CRC) by tricarboxylic cycle intermediates such as succinate. Total phospholipase (PLC) activity in osmoprotectant-induced cultures of P. aeruginosa PAO1 supplemented with 20 mM succinate was three- to fourfold lower than the levels in cultures supplemented with the non-catabolite-repressive substrate lactate. Analyses of osmoprotectant-dependent plcH expression in a derivative of strain PAO1 containing a plcH::lacZ operon fusion showed that (i) succinate prevented induction of plcH expression by osmoprotectants; and (ii) addition of succinate reduced or shut down further expression of plcH in osmoprotectant-induced bacteria, while cultures supplemented with lactate had little or no change in plcH expression. RNase protection analysis confirmed that repression of plcH occurs at the transcriptional level. However, a P. aeruginosa mutant decoupled in CRC exhibited a phenotype similar to that of the wild-type strain (PAO1) with respect to succinate-dependent repression of plcH expression. Osmoprotectant-induced total PLC activities, levels of expression of plcH measured with the same plcH::lacZ fusion, and levels of plcH transcription in a CRC-deficient strain reflected those seen in strain PAO1. This indicates that CRC of plcH functions by a distinct mechanism which differs from that regulating the glucose or mannitol catabolic pathway. A strain carrying a mutation in vfr, which encodes the Escherichia coli Crp homolog in P. aeruginosa, still exhibited a wild-type phenotype with respect to osmoprotectant-dependent expression and CRC of plcH. These data indicate that there is a novel CRC system that regulates the expression of plcH in P. aeruginosa.

In VivoInterference of Paromomycin with Mitochondrial Activity of Leishmania

Paromomycin is an aminocyclitol aminoglycoside antibiotic used for the treatment of leishmaniasis. In view of the central role of mitochondria in cellular energetics and metabolism, its effect on in vivomitochondrial activities of Leishmania donovanipromastigotes—the parasite flagellate form—was investigated. The approach used flow cytometry, amperometric measure of O 2consumption, and, as a global estimate of mitochondrial dehydrogenases, thiazolyl blue reduction (MTT test); some in vitrocontrols were also made. When added to promastigote cultures for 24–72 h at 150–200 μ M(= LC 50), paromomycin doubled the generation time, inhibited respiration, and lowered its associated electric potential difference across mitochondrial membranes, as measured by rhodamine 123 fluorescence. The chemical analogue neomycin was ineffective. Furthermore, the in vivomitochondrial dehydrogenase activities were lower, seemingly because of the shortage of respiratory substrates. Indeed, succinate addition to paromomycin-treated cultures partly restored mitochondrial membrane potential. However, no immediate effect of paromomycin on respiration was observed, neither inhibition of redox chain nor increase of membrane permeability (uncoupling). It is proposed that paromomycin acts at a metabolic level upstream of the respiratory chain itself. This would have the observed delayed consequence because the cell energy supply would progressively decline since it depends upon the proton gradient—viz., membrane potential—generated by respiration. In conclusion, paromomycin is an antibiotic affecting the cell's energetic metabolism; the respiratory dysfunction it induces may be a crucial aspect of its action against Leishmaniaand possibly other cells.

Reversibility of thiamine deficiency-induced partial necrosis and mitochondrial uncoupling by addition of thiamine to neuroblastoma cell suspensions.

Culture of neuroblastoma cells in the presence of low thiamine concentration (16 nM) and of the transport inhibitor amprolium leads to the appearance of signs of necrosis: the chromatin condenses, the oxygen consumption decreases and is uncoupled, the mitochondrial cristae are disorganized, the thiamine diphosphate-dependent dehydrogenase activities are impaired. When 10 microM thiamine are added to these cells, the basal respiration increases, the coupled respiration is restored and mitochondrial morphology is recovered within 1 h. Addition of succinate, which is oxidized via a thiamine diphosphate-independent dehydrogenase, to digitonin-permeabilized cells immediately restores a coupled respiration. Our results suggest that the slowing of the citric acid cycle is the cause of the biochemical lesion induced by severe thiamine deficiency and that part of the mitochondria remain functional.

Rapid uncoupling of oxidative phosphorylation accompanies glutamate toxicity in rat cerebellar granule cells.

A 100 microM glutamate pulse administered to rat cerebellar granule cells causes a very rapid and progressive decrease in both cell and mitochondrial oxygen consumption caused by glucose and succinate addition, respectively. The respiratory control ratio, which reflects the ability of mitochondria to produce ATP, is reduced by 50% within the first 30 min after glutamate addition. Subsequent to glutamate exposure, a progressive decrease of respiratory control ratio to almost 1 was found within the following 3-5 h. The addition of extra calcium had no effect per se on oxygen consumption by cell homogenate.

Catalytic enzyme histochemistry and biochemical analysis of dihydroorotate dehydrogenase/oxidase and succinate dehydrogenase in mammalian tissues, cells and mitochondria.

Dihydroorotate dehydrogenase (EC 1.3.3.1 or EC 1.3.99.11) catalyzes the fourth sequential step in the de novo synthesis of uridine monophosphate. In eukaryotes it is located in the inner mitochondrial membrane, with ubiquinone as the proximal and cytochrome oxidase as the ultimate electron transfer system, whereas the rest of pyrimidine biosynthesis takes place in the cytosol. Here, the distribution of dihydroorotate dehydrogenase activity in cryostat sections of various rat tissues, and tissue samples of human skin and kidney, was visualized by light microscopy using the nitroblue tetrazolium technique. In addition, a hydrogen peroxide-producing oxidase side-reactivity of dihydroorotate dehydrogenase could be visualized by trapping the peroxide with cerium-diaminobenzidine. The pattern of activity was similar to that of succinate dehydrogenase, but revealed a less intensive staining. High activities of dihydroorotate dehydrogenase were found in tissues with known proliferative, regenerative, absorptive or excretory activities, e.g., mucosal cells of the ileum and colon crypts in the gastrointestinal tract, cultured Ehrlich ascites tumor cells, and proximal tubules of the kidney cortex, whilst lower activities were present in the periportal area of the liver, testis and spermatozoa, prostate and other glands, and skeletal muscle. Dihydroorotate dehydrogenase and succinate dehydrogenase activity in Ehrlich ascites tumor cells grown in suspension culture were quantified by application of nitroblue tetrazolium or cyanotolyl tetrazolium and subsequent extraction of the insoluble formazans with organic solvents. The ratio of dihydroorotate dehydrogenase to succinate dehydrogenase activity was 1:4. This was in accordance with that of 1:5 obtained from oxygen consumption measurement of isolated mitochondria on addition of dihydroorotate or succinate. The ratio determined with mitochondria from animal tissues was up to 1:15 (rat liver, bovine heart). The application of the enzyme inhibitors brequinar sodium and toltrazuril verified the specificity of the histochemical and biochemical methods applied.

Propionate modulation of ruminal ketogenesis.

The ruminal epithelium is the primary source, through the metabolism of butyrate, of circulating ketone bodies in fed ruminants. Volatile fatty acid metabolism was investigated in short-term (2-h) incubations of isolated sheep ruminal epithelial cells. Ruminal epithelial cells were isolated from ruminal papillae via serial tryptic digestion. Cells were incubated in the presence of various combinations of butyrate (0, .5, 1, 5, 10, or 25 mM), propionate (0, .1, .5, 1, 5, 10, 25, 50, or 100 mM), acetate (0, .5, 1, 5, 25, 50, or 100 mM), and succinate (0, 5, 15, 50 mM) to evaluate the effects of these substrates on butyrate metabolism. Variables measured included beta-hydroxybutyrate (beta-HBA) and acetoacetate (AcAc) production from butyrate, [14C]butyrate oxidation to 14CO2, and lactate and pyruvate formation from propionate and succinate. Butyrate oxidation to CO2, beta-HBA and total ketones produced (nanomoles of butyrate metabolized per 10(6) cells per minute) were linear over the 2-h incubation period (y = .805X + 2.24, r2 = .78; y = 1.02X - 9.1, r2 = .72; and y = 2.238X - 11.76, r2 = .65, respectively). Acetate inhibited beta-HBA formation from butyrate (P < .05) when present at concentrations greater than 5 mM, although a dose-dependence was not consistently exhibited. However, butyrate oxidation to beta-HBA was stimulated (P < .05) by propionate while acetoacetate production declined (P < .05), resulting in no net change in ketone body production (P > .05). A concomitant increase in lactate and pyruvate production was noted with increasing concentrations of propionate. Succinate addition also increased lactate production by the ruminal epithelial cells. In contrast to propionate, succinate addition resulted in a decrease in beta-HBA formation from butyrate (P < .05). Butyrate metabolism by isolated ruminal epithelial cells is influenced by other VFA produced in the rumen, and propionate-induced stimulation of beta-HBA from butyrate does not seem to be mediated via succinate but rather is a result of a shift in the mitochondrial NADH/NAD status.

Endogenous ubiquinol prevents protein modification accompanying lipid peroxidation in beef heart submitochondrial particles

This article is a study of the relationship between lipid peroxidation and protein modification in beef heart submitochondrial particles, and the protective effect of endogenous ubiquinol (reduced coenzyme Q) against these effects. ADP-Fe 3· and ascorbate were used to initiate lipid peroxidation and protein modification, which were monitored by measuring TBARS and protein carbonylation, respectively. Endogenous ubiquinone was reduced by the addition of succinate and antimycin. The parameters investigated included extraction and reincorporation of ubiquinone, and comparison of the effect of ubiquinol with those of various antioxidant compounds and enzymes, as well as the iron chelator EDTA. Under all conditions employed there was a close correlation between lipid peroxidation and protein carbonylation, and the inhibition of these effects by endogenous ubiquinol. SDS-PAGE analysis revealed a differential effect on individual protein components and its prevention by ubiquinol. Conceivable mechanisms behind the observed oxidative modifications of membrane phospholipids and proteins and of the role of ubiquinol in preventing these effects are considered.

Ca(2+)-induced mitochondrial membrane permeabilization: role of coenzyme Q redox state.

Rotenone-poisoned rat liver mitochondria energized by succinate addition, after a 5-min period of preincubation in presence of 10 microM Ca2+, produce H2O2 at much faster rates, undergo extensive swelling, and are not able to retain the membrane potential and accumulated Ca2+. Similar results were obtained when a suspension of rat liver mitochondria preincubated in anaerobic medium for 5 min was reoxygenated. The addition of either ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, ruthenium red, catalase, or dithiothreitol, just before succinate or O2 addition, prevented mitochondrial swelling, indicating the involvement of Ca2+, reactive oxygen species, and oxidation of membrane protein thiols in this process of membrane permeabilization. Inhibition of mitochondrial swelling by cyclosporin A suggests that the membrane alterations observed under these experimental conditions are related to opening of the permeability transition pore. The presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone, which prevents Ca2+ cycling across the membrane, did not inhibit mitochondrial swelling when Ca2+ influx into the mitochondrial matrix was driven by a high Ca2+ gradient. When rotenone plus antimycin A-poisoned mitochondria were energized by N,N,N',N'-tetramethyl-p-phenylenediamine, which reduces respiratory chain complex IV, mitochondrial swelling did not occur, unless succinate, which reduces coenzyme Q, was also added. It is concluded that reduced coenzyme Q is the electron source for oxygen radical production during Ca(2+)-stimulated oxidative damage of mitochondria.

Biodegradation of 2-chloroethanol by freely suspended and adsorbed immobilized Pseudomonas putida US2 in soil.

The degradation of 2-chloroethanol by Pseudomonas putida US2 was investigated in batch, repeated batch and continuous cultures especially in a packed-bed fermenter with sand. The degradation of 2-chloroethanol was connected with a release of protons, which led to a decrease of the pH in the medium. Higher initial concentration than 25 mM 2-chloroethanol were not degraded completely because they entailed a decrease of the pH to 5.0, which inhibited further growth and degradation. P. putida US2 showed a typical repression of catabolites and diauxic growth with succinate as cosubstrate. The addition of succinate as a second substrate caused a decrease in degradation of 2-chloroethanol. Activated sludge added to adsorbed cultures in a continuous fermentation did not lead to a decrease in metabolic activity. After 2 weeks of continuous cultivation the specialized strain could be retained.

Regulatory effects of exogenous branched-chain amino acids in Nicotiana plumbaginifolia cell suspension cultures

Nicotiana plumbaginifolia suspension cultured cells were grown on medium supplemented with valine, leucine and isoleucine, singly or in combination. The effects of the three branched-chain amino acids on cell growth rate and on the activity of acetohydroxyacid synthase (AHAS), the first enzyme (and the main regulative site) of their biosynthetic pathway, were studied. Results showed that valine and leucine, at concentrations ranging from 10−4 to 10−3M, inhibit growth, and at higher doses (from 10−2 to 10−1M) AHAS activity. Growth, but not AHAS activity, was affected also by isoleucine. The addition of ammonium succinate to the culture medium, in order to counteract a possible general inhibitory effect of these compounds on nitrogen metabolism, relieved only partially their cytotoxicity. Feeding cells with equimolar mixtures of the three amino acids resulted in a minor but reproducible decrease in AHAS level, which was proportional to the dose. A similar result was obtained also on N. plumbaginifolia seedlings, suggesting that in this species a modulation of enzyme level could play a role in controlling the flow of metabolites through the pathway.

The Regulation of Pyruvate Dehydrogenase Activity in Pea Leaf Mitochondria (The Effect of Respiration and Oxidative Phosphorylation).

The regulation of the pea (Pisum sativum) leaf mitochondrial pyruvate dehydrogenase complex by respiratory rate and oxidative phosphorylation has been investigated by measuring the respiratory activity, the redox poise of the quinone pool (Q-pool), and mitochondrial pyruvate dehydrogenase (mtPDC) activity under various metabolic conditions. It was found that, under state 4 conditions, mtPDC activity was unaffected by either the addition of succinate, 2-oxoglutarate, or glycine or the overall respiratory rate and redox poise of the Q-pool but was partially inhibited by NADH due to product inhibition. In the presence of ADP significant inactivation of PDC, which was sensitive to oligomycin, was observed with all substrates, apart from pyruvate, suggesting that inactivation was due to ATP formation. Inactivation of PDC by ADP addition was observed even in the presence of carboxyatractyloside, an inhibitor of the ATP/ADP translocator, suggesting that other mechanisms to facilitate the entry of adenylates, in addition to the adenylate carrier, must exist in plant mitochondria.

Family of glycosyl transferases needed for the synthesis of succinoglycan by Rhizobium meliloti

Rhizobium meliloti produces an acidic exopolysaccharide, termed succinoglycan or EPS I, that is important for invasion of the nodules that it elicits on its host, Medicago sativa. Succinoglycan is a high-molecular-weight polymer composed of repeating octasaccharide subunits. These subunits are synthesized on membrane-bound isoprenoid lipid carriers, beginning with a galactose residue followed by seven glucose residues, and modified by the addition of acetate, succinate, and pyruvate. Biochemical characterizations of lipid-linked succinoglycan biosynthetic intermediates from previously identified exo mutant strains have been carried out in our laboratory (T. L. Reuber and G. C. Walker, Cell 74:269-280, 1993) to determine where each mutation blocks the biosynthetic pathway. We have carried out a fine structure genetic analysis of a portion of the cluster of exo genes present on the second symbiotic megaplasmid of R. meliloti and have identified several new genes. In addition, the DNA sequence of 16 kb of the exo cluster was determined and the genetic map was correlated with the DNA sequence. In this paper we present the sequence of a family of glycosyl transferases required for the synthesis of succinoglycan and discuss their functions.

Substrate-stimulated oxygen consumption by isolated rat brain microvessels in the presence and absence of ATP.

We report here properties of isolated brain microvessels such as the rate of oxygen consumption with different substrates; the permeabilizing effect of added ATP is studied. With the isolation procedure presented the cerebral endothelium has a metabolic activity comparable to that reported in the literature. The respiratory rate of the microvessels is not affected by the addition of ATP, whereas it is significantly increased by addition of succinate and alpha-chetoglutarate. The exposure of the isolated brain capillaries to ATP, in a Ca(2+)-free medium, increases the uptake of 6-carboxyfluorescein. This may be due to pores opened by ATP in the endothelial cell membrane in the absence of divalent cations.

Allylamine toxicity in embryonic myocardial myocyte reaggregate cultures: The role of extracellular metabolism by benzylamine oxidase

The cardiotoxicity of allylamine (AA) is considered to be mediated by metabolism to the highly reactive aldehyde, acrolein (ACR). The toxicity of AA to myocardial myocyte reaggregate cultures (MMR) was assessed by measuring percentage beating as a marker of functional viability. The studies demonstrated that AA toxicity could be prevented by inhibitors of benzylamine oxidase (BZO) but not by inhibitors of monoamine oxidase A or B. Addition of exogenous BZO markedly potentiated the toxicity, an effect that could be blocked by semicarbazide, an inhibitor of BZO. The present studies support the view that AA is metabolized by BZO to the proximate toxicant, ACR. In serum-free cultures, high concentrations (0.5–2.5 m m) of AA were required to cause any loss of viability, and as the concentration of serum in the medium was increased so the loss of viability was induced by AA; in MMR maintained in 50% foetal calf serum, all viability was lost after 3 hr of exposure to 100 μ m-AA. Mercaptoethanesulphonate (MESNA), a scavenger of reactive species that is known not to penetrate myocytes, prevented the toxicity both of 100 μ m-AA and 100 μ m-ACR to MMR in serum-supplemented medium. In contrast, when MMR in serum-free medium were exposed to high concentrations of AA, MESNA had no moderating effect. These findings suggest that AA undergoes extracellular metabolism to ACR in serum-supplemented medium because of the presence of BZO in serum. It is clear that extracellular metabolism is of great importance in the pathogenesis of AA-induced toxicity to MMR in serum-supplemented medium. The toxicity of AA was also prevented by the iron chelator desferrioxamine (DF) at a concentration shown not to inhibit significantly the activity of BZO. ACR toxicity too, was inhibited by DF. This suggests a role for free radicals in the toxicity of AA to MMR, as DF chelates iron, thus preventing the catalysis of free radical reactions (Halliwell and Gutteridge, 1986). Addition of α-tocopherol succinate, an inhibitor or lipid peroxidation, to the cultures also reduced the toxicity of AA, which provides some evidence of the role of lipid peroxidation in the mechanism of AA toxicity to MMR. That process too, commonly involves free radical mechanisms. The results are discussed with reference to the action of AA in vivo, in order to consider whether extracellular metabolism might be of importance in the mechanism of toxicity to the whole animal.

Palmitate metabolism by isolated sheep rumen epithelial cells

Ruminal palmitate metabolism was examined using an isolated cell system. Palmitate oxidation to 14CO2 by rumen epithelial cells isolated from the rumens of mature sheep was linear during the course of a 2-h incubation (11.1 nmoles.million cells-1.2 h-1) and 3.6 times the rate of palmitate oxidation by cells isolated from neonatal rumen (3.1 nmoles.million cells-1.min-1). Subsequent experiments were conducted with mature rumen epithelial cells. Neither acetate (50 mM), propionate (10 mM), dibutyryl cAMP (.2 mM), nor insulin (10 mU/mL) altered palmitate oxidation to CO2. However, butyrate (10 mM) addition reduced (P less than .05), and ammonia (15 mM) tended to reduce (P less than .10), palmitate oxidation (51.6 and 82.0% of control, respectively), whereas addition of glucose (2.5 mM) increased (P less than .05) palmitate oxidation (151% of control). Of the compounds tested, only propionate, butyrate, and ammonia reduced palmitate oxidation to total acid-soluble metabolites. Propionate (10 mM) addition completely abolished palmitate oxidation to acid-soluble metabolites. Succinate addition (5 to 50 mM) increased palmitate oxidation to CO2 but exhibited no consistent effect on palmitate oxidation to either acid-soluble metabolites or beta-hydroxybutyrate. Propionate completely abolished palmitate oxidation to beta-hydroxybutyrate, suggesting that propionate-induced inhibition of palmitate oxidation is not mediated via succinate. The data indicate 1) that rumen epithelium is capable of oxidizing palmitate, 2) that ruminal palmitate oxidation may be subject to regulation by developmental factors, and 3) that palmitate metabolism seems to be influenced more by ruminally derived metabolites than by factors derived exclusively from the general circulation.

Growth yield increase and ATP formation linked to succinate decarboxylation in Veillonella parvula.

Veillonella parvula strain 259 (= DSM 2007) was able to grow on a mineral salts medium supplemented with (per litre) 1 g yeast extract, 1 g Tween-80, and 3 mg putrescine.2 HCl, with 6 mM thioglycolate as reductant and lactate as growth substrate. Succinate did not serve as a growth substrate, but when added in conjunction with lactate, it was decarboxylated to propionate and resulted in a measurable increase in growth yield, corresponding to the formation of 2.4 g cell dry mass per mol succinate. A growth yield increase linked to succinate metabolism occurred only while lactate was also being metabolised. Experiments with cell suspensions showed that succinate decarboxylating activity was constitutive. Addition of succinate produced clear increases in cellular ATP levels in ATP-depleted washed cells.

In Vitro Drug Sensitivity of Cells From Children With Leukemia Using the MTT Assay With Improved Culture Conditions

The knowledge about drug resistance in childhood leukemias and acute lymphoblastic leukemia (ALL) in general is limited. This is because of the lack of a suitable in vitro drug sensitivity assay, which is in part due to low in vitro ALL cell survival. We recently adapted the highly efficient 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay to test cells from ALL patients and showed that its results were comparable with those of the DiSC assay, up to now the most valid but laborious assay. In this study, in vitro drug sensitivity was assessed in cells from 82 children with leukemia, 79 of whom had ALL, with the MTT assay. Dose response curves were obtained for 6-mercaptopurine, 6-thioguanine (6-TG), prednisolone (Pred), daunorubicin (DNR), vincristine (VCR), cytosine arabinoside (Ara-C), L-asparaginase (L-Asp), mafosfamide, and mustine. A cytotoxic effect of methotrexate could be detected in only a few cases. Large interindividual differences in drug sensitivity were detected. Compared with leukemia cells from newly diagnosed patients, leukemia cells from relapsed patients were significantly more in vitro resistant to 6-TG, Pred, Ara-C, mafosfamide and mustine but not to DNR, VCR, and L-Asp. Improvements of culture medium and methods to increase MTT reduction were studied. From 10 components tested, addition of insulin and bovine serum albumin to serum-containing medium improved ALL cell survival. Addition of succinate did not increase the amount of MTT reduction. We conclude that the in vitro MTT assay highly facilitates large-scale studies on drug resistance of ALL patients that can lead to rational improvements in existing treatment protocols.