Regulation of carbon and electron flow in Clostridium acetobutylicum grown in chemostat culture at neutral pH on mixtures of glucose and glycerol.

The metabolism of Clostridium acetobutylicum was manipulated, at neutral pH and in chemostat culture, by changing the overall degree of reduction of the substrate, using mixtures of glucose and glycerol. Cultures grown on glucose alone produced only acids, and the intracellular enzymatic pattern indicated the absence of butyraldehyde dehydrogenase activity and very low levels of coenzyme A-transferase, butanol, and ethanol dehydrogenase activities. In contrast, cultures grown on mixtures of glucose and glycerol produced mainly alcohols and low levels of hydrogen. The low production of hydrogen was not associated with a change in the hydrogenase level but was correlated with the induction of a ferredoxin-NAD reductase and a decreased level of NADH-ferredoxin reductase. The production of alcohols was related to the induction of a NAD-dependent butyraldehyde dehydrogenase and to higher expression of NAD-dependent ethanol and butanol dehydrogenases. The coenzyme A-transferase was poorly expressed, and thus no acetone was produced. These changes in the enzymatic pattern, obtained with cultures grown on a mixture of glucose and glycerol, were associated with a 7-fold increase of the intracellular level of NADH and a 2.5-fold increase of the level of ATP.     

Proteins of the Inner Membrane of Escherichia coli: Changes in Composition Associated with Anaerobic Growth and Fumarate Reductase Amber Mutation

The inner membrane fractions of Escherichia coli grown anaerobically and aerobically were isolated, and their proteins were compared by electrophoresis in polyacrylamide gels. To maximimize the differences between the preparations, the anaerobic cultures were grown on complex medium with added glucose, but glucose was omitted from the aerobic cultures to prevent catabolite repression. The pattern of bands in the two types of preparation differed considerably, and changes in approximately 20 components were observed. In particular, the band identified as succinate dehydrogenase in aerobic preparations was greatly reduced in anaerobic preparations. Mutants lacking fumarate reductase were isolated, and inner membrane preparations of an frd amber mutant were deficient in a major component of 75,000 daltons and possibly a minor one of 87,500 daltons. The former was also present in greater amounts in anaerobic preparations and could represent a fumarate reductase subunit.     

Carbohydrate metabolism enzymatic activity and its alteration under the influence of thyroid hormone during tumor growth]

The activity of hexokinase (HK), its isoenzymes, glucose-6-phosphatase and glucose-6-phosphate dehydrogenase, and the triiodothyronine (T3) effect on this activity in the liver tissue of mice bearing transplantable hepatoma 22a were studied in different periods of the tumor growtn. It was shown that alterations in the activity of the enzymes in the liver of tumor-bearing mice occurred already in the presence of a small tumor. More profound alterations in the activity of all enzymes studied, apart from those in the enzymatic pattern of HK, could be observed starting from day 21after the tumor transplantation. In the initial stages of the hepatoma growth the activity of the test enzymes in the liver was regulated by thyroid hormone. The effect of Ta on the activity of the enzymes in the host liver was gradually lost in the course of the tumor growth.     

Chinese hamster monomeric carbonyl reductases of the short-chain dehydrogenase/reductase superfamily

Chinese hamster monomeric carbonyl reductases (CHCRs) belong to the short-chain dehydrogenase/reductase (SDR) superfamily, which is a family of enzymes that metabolize many endogenous and xenobiotic compounds. We previously cloned three carbonyl reductase cDNAs-Chcr1, Chcr2, and Chcr3. By performing spectrophotometric analyses, we indicated that the enzymes CHCR1, CHCR2, and CHCR3 had similar specificities toward steroids; only CHCR3 did not show any reactivity with prostaglandins (PGs). In the present study, we investigated the characteristics of CHCRs in detail, that is, the differences in their expression patterns, physicochemical properties, and enzymatic activities. CHCR1 exhibited sex-dependent expression patterns. CHCRs showed multiple surface potentials in the zeta potential analysis and CHCR3 exhibited an isatin reductase activity with a high K(m) value. By the present HPLC-analysis, all the three enzymes exhibited PGF(2alpha) dehydrogenase activity and could oxidize PGF(2alpha) to PGE(2) and 15-keto-PGF(2alpha), i.e., the three enzymes exhibited 9- and 15-hydroxy PG dehydrogenase activities. Moreover, 15-keto-PGE(2) was detected in a comparatively higher amount in the dehydrogenase reaction products of CHCR2 than in those of CHCR1 and CHCR3, suggesting that CHCR2 can oxidize PGE(2) and/or 15-keto-PGF(2alpha) to 15-keto-PGE(2); however, these two PGs did not seem to be efficient substrates of CHCR1. Despite the differences in the dehydrogenase activities between CHCR1 and CHCR2, PGE(2) reductase activities of the two enzymes were similar, and PGF(2alpha) was predominantly produced from PGE(2) as a result of the PG 9-keto reductase activity. On the other hand, CHCR3 exhibited a reduced PGE(2) reductase activity. In conclusion, although the CHCRs share a high degree of sequence identity (>70%), they clearly differed in their enzymatic characteristics.     

The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase and the rate of sterol synthesis diminish in cultures with high cell density

The specific activity of HMG-CoA reductase, the major rate-limiting enzyme in the sterol biosynthetic pathway, declined linearly with increasing cell density in four different lines of mammalian cell cultures. As expected, this caused the rates of sterol synthesis from [14C]acetate to decline in a parallel manner. The decrease in reductase activity in the dense cultures was also correlated with decreased incorporation of [14C]acetate into fatty acids and [3H]thymidine into DNA. In contrast, the activities of two enzymes, NADH dehydrogenase and 5'-nucleotidase, which are not involved in lipid synthesis, were independent of changes in cell density. The simplest explanation for these data is tht HMG-CoA reductase and the synthesis of sterol and fatty acids are regulated in concordance with the rate of cell growth and proliferation.     

Polyol pathway along the bovine epididymis

During the epididymal transit, male gametes acquire new surface proteins necessary for their fertilizing ability. We have previously shown that membranous vesicles, called epididymosomes, interact with sperm surface within the epididymal fluid allowing transfer of some proteins to different subcellular compartments of spermatozoa. We previously showed that one of the major proteins associated with epididymosomes was an aldose reductase (gene: AKR1B5) and confirmed that aldose reductase is located in the epithelial cells bordering the intraluminal compartment of the epididymis. The present study shows that cytosolic aldose reductase activity was maximal in the proximal and middle segments of the epididymis and decreased in the distal epididymis. Western and Northern blot analysis confirmed the distribution pattern of aldose reductase and of the encoding mRNA. The optimal pH of epididymal aldose reductase was 6.0-6.5 when glucose was used as a substrate; this corresponds to the pH of the intraluminal epididymal fluid. In order to evaluate the possible involvement of sorbitol in sperm physiology, Western blot of tissue homogenates were probed with an anti-sorbitol dehydrogenase antibody. The amount of enzyme immunodetected was higher in the proximal and distal segments of the epididymis when compared to the amount detectable in the middle segment of the epididymis. Sorbitol dehydrogenase activity as well as the level of the encoding mRNA showed the same pattern of distribution. Furthermore, immunohistological studies using the anti-sorbitol dehydrogenase revealed that this enzyme was synthesized by the epididymal epithelial cells bordering the intraluminal compartment. Knowing the importance of sorbitol and fructose in sperm metabolism, we hypothesized that the polyol pathway is involved in the modulation of sperm motility within the epididymis.     

Mitochondrial development during myogenesis.

The enzymatic and ultrastructural pattern of mitochondrial differentiation was investigated during myogenesis. Succinate cytochrome C reductase (SCR), a mitochondrial enzyme complex, increased in activity in developing chick thigh muscle in vivo and in vitro. SCR increase in vitro occurred subsequent to myoblast fusion and correlated with the period of increasing creatine phosphokinase (CPK) activity. Fusion-arrest and Ca²⁺-reversal experiments indicated an apparent coordination between CPK and SCR enzymatic increases and fusion. Analysis of SCR activity in fibroblast cultures suggested that the enzymatic increases observed in differentiating muscle cultures reflected myocyte differentiation, rather than fibroblast contamination or a unique property of the tissue culture environment. Morphological transitions in the myogenic mitochondria were temporally correlated with increased SCR activity. During myogenesis, the mitochondria enlarged in length and volume, exhibited an increase in matrix density, oriented in parallel to the long axis of the myofibrils, and contained increased numbers of parallel cristae. Many mitochondria in fusion-inhibited muscle cultures resembled those found in prefusion myoblasts, although mature mitochondria were observed in some fusion-blocked cells. Quantitative stereological analyses of these mitochondrial changes parallel the biochemical data and suggest that ultrastructural and enzymatic changes in the mitochondria are an integral part of myodifferentiation.     

Glucose-6-phosphate dehydrogenase activity and NADPH/NADP+ ratio in liver and pancreas are dependent on the severity of hyperglycemia in rat

Hyperglycemia is associated with metabolic disturbances affecting cell redox potential, particularly the NADPH/NADP+ ratio and reduced glutathione levels. Under oxidative stress, the NADPH supply for reduced glutathione regeneration is dependent on glucose-6-phosphate dehydrogenase. We assessed the effect of different hyperglycemic conditions on enzymatic activities involved in glutathione regeneration (glucose-6-phosphate dehydrogenase and glutathione reductase), NADP(H) and reduced glutathione concentrations in order to analyze the relative role of these enzymes in the control of glutathione restoration. Male Sprague-Dawley rats with mild, moderate and severe hyperglycemia were obtained using different regimens of streptozotocin and nicotinamide. Fifteen days after treatment, rats were killed and enzymatic activities, NADP(H) and reduced glutathione were measured in liver and pancreas. Severe hyperglycemia was associated with decreased body weight, plasma insulin, glucose-6-phosphate dehydrogenase activity, NADPH/NADP+ ratio and glutathione levels in the liver and pancreas, and enhanced NADP+ and glutathione reductase activity in the liver. Moderate hyperglycemia caused similar changes, although body weight and liver NADP+ concentration were not affected and pancreatic glutathione reductase activity decreased. Mild hyperglycemia was associated with a reduction in pancreatic glucose-6-phosphate dehydrogenase activity. Glucose-6-phosphate dehydrogenase, NADPH/NADP+ ratio and glutathione level, vary inversely in relation to blood glucose concentrations, whereas liver glutathione reductase was enhanced during severe hyperglycemia. We conclude that glucose-6-phosphate dehydrogenase and NADPH/NADP+ were highly sensitive to low levels of hyperglycemia. NADPH/NADP+ is regulated by glucose-6-phosphate dehydrogenase in the liver and pancreas, whereas levels of reduced glutathione are mainly dependent on the NADPH supply.     

Changes induced by aging and drug treatment on cerebral enzymatic antioxidant system

The age-related modifications of the participants to the cerebral enzymatic antioxidant system (superoxide dismutase, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase) were evaluated in four brain regions from male Wistar rats aged 5, 10, 15, 20, 25, 30, and 35 months. Both the specific enzyme activity and the profile of any enzyme tested markedly differ with age according to the region examined: parieto-temporal cortex, caudate-putamen, substantia nigra and thalamus. This inhomogeneous age-related profile of enzyme activities could explain both the controversial data of literature and the different regional vulnerability of the brain tissue to damage with aging. In rats aged 10, 20, or 30 months, the chronic i.p. treatment for two months with papaverine or ergot alkaloids (dihydroergocristine, dihydroergocornine, dehydroergocriptine) suggests that the antioxidant enzyme activities may be influenced according to the agent utilized, the brain region tested, and the age of the animal. In any case, small differences in the drug structure support marked differences in the type and extent of the intervention on the antioxidant enzymatic system.     

Enzyme reaction rate studies in electromotor neurons of the weakly electric fish Apteronotus leptorhynchus

A histochemical analysis of reaction rates of a series of enzymes was performed in electromotor neurons of the weakly electric fish Apteronotus leptorhynchus. These neurons were selected because of their functional homogeneity. The high metabolic activity of these cells as well as their large size facilitate cytophotometric analysis in cryostat sections. Sections were incubated for the activity of hexokinase, glucose-6-phosphate dehydrogenase, succinate dehydrogenase, NADPH dehydrogenase, NADPH ferrihaemoprotein reductase and beta-hydroxybutyrate dehydrogenase. All media contained polyvinyl alcohol as tissue stabilizer and Nitro BT as final electron acceptor. Measurements were performed with a Vickers M85a cytophotometer. Linear relationships between the specific formation of formazan (test minus control reaction) and incubation time were obtained for all enzymes although some reactions showed an initial lag phase or an intercept with the ordinate. The relatively high activities of hexokinase, succinate dehydrogenase and the extremely low activity of hydroxybutyrate dehydrogenase indicate that energy is mainly supplied by glycolysis. Glucose-6-phosphate dehydrogenase showed a high activity whereas NADPH reductase and dehydrogenase activity were low in electromotor neurons, indicating that the NADPH generated is largely used for biosynthesis. Despite their synchronous firing pattern activity, electromotor neurons showed a considerable heterogeneity with respect to their metabolic activity.     

Regulation of the NADH and NADPH-ferredoxin oxidoreductases in clostridia of the butyric group.

NADH and NADPH-ferredoxin oxidoreductases have been studied in Clostridium acetobutylicum, Cl. tyrobutyricum and Cl. pasteurianum. The study of the distribution and regulation of these enzymatic activities in well-defined culture conditions, reveals that the essential function of NADPH-ferredoxin oxidoreductase is to produce NADPH, while NADH-ferredoxin oxidoreductase can, depending on cellular conditions, produce or oxidize NADH. When these Clostridia use glycolysis, regulation of the NADH-ferredoxin oxidoreductase by acetyl-CoA (obligatory activator of NADH-ferroxin reductase activity) and by NADH (competitive inhibitor of ferredoxin-NAD+ reductase activity) allow the enzymes to function correlatively with glyceraldehyde-3-phosphate dehydrogenase and thus control the levels of NAD+ and NADH in the cell. In Cl. tyrobutyricum and Cl. pasteurianum, the ferredoxin-NADP+ reductase activities are regulated by NAD+ and NADH in accordance with the intracellular concentrations of these coenzymes. In Cl. tyrobutyricum growing on pyruvate/acetate, NADH and NADPH-ferredoxin reductase activities cannot be detected; only the ferredoxin-NAD+ and ferredoxin-NADP+ reductase activities are found. In this Clostridium, regulation of the ferredoxin-NADP+ reductase activity is the same whether it is grown on glucose or pyruvate. Contrary to this, the ferredoxin-NAD+ reductase activity undergoes a drastic change, since NADH no longer controls the enzymatic activity. In this case regulation is no longer necessary, since glyceraldehyde-3-phosphate dehydrogenase does not function.     

Enzyme activities of the primary and secondary metabolism of simultaneously permeabilized and immobilized plant cells

After various permeabilization procedures, plant cells obtained from suspension cultures of Catharanthus roseus are permeable to enzyme substrates which cannot enter the intact cell. Five enzymes of the primary metabolism, hexokinase, glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, malic enzyme, and citrate dehydrogenase, are studied with special emphasis on the two-enzyme system hexokinase/glucose-6-phosphate dehydrogenase. It is found that permeabilized cells immobilized in agarose retain their enzymatic activities far longer on storage than nonimmobilized cells. Whereas cells permeabilized by various methods show different initial enzymatic activity, the subsequent decrease of activity with time is at the same relative rate. Optimal initial activity is found with dimethyl sulfoxide-treated plant cells. As an enzyme of the secondary metabolism, we choose cathenamice reductase forming ajmalicine alkaloids from cathenamine. It is found that in dimethyl sulfoxide-treated cells the enzyme activity remains intact and that the addition of the coenzyme required in this step, NADPH, considerably increases the yield of product formed. Also, excretion into the medium is enhanced in both these immobilized and permeabilized systems.     

Comparative impact of cephaloridine on glutathione and related enzymes in LLC-PK1, LLC-RK1, and primary cultures of rat and rabbit proximal tubule cells

Among kidney tubular epithelial cell types, proximal tubule cells are one of the major renal targets for xenobiotics. Several in vitro culture models have been proposed for use of proximal tubule cells for in vitro pharmacotoxicology studies. This paper reports a comparative study of the response to cephaloridine exposure of two established cell lines from pig (LLC-PK1) and rabbit (LLC-RK1) kidneys and primary cultures of rat and rabbit proximal tubule cells. These cultured cells were first compared for their levels of activity of -methylglucopyranoside transport, alkaline phosphatase, succinate dehydrogenase, and NADPH cytochrome c reductase, their glutathione-dependent activity levels, and their adenylate cyclase response pattern to stimulation by PTH and AVP. The results presented show major phenotypic differences between these four cellular models. The differences observed in glutathione-dependent mechanism activities and regulation may in part be responsible for the variability of the responses of these four cellular models when exposed to cephaloridine.Abbreviations AVP arginine vasopressin - GGT -glutamyl transpeptidase - GRED glutathione reductase - GSH glutathione - GST glutathione S-transferase - PTC proximal tubule cells - PTH parathyroid hormone - SDH succinate dehydrogenase     

Quantitative histochemistry of the sorbitol pathway in glomeruli and small arteries of human diabetic kidney.

Recent evidence has suggested a role for the polyol pathway in pathogenesis of cell damage in diabetes Glucose may be phosphorylated to glucose-6-phosphate via hexokinase and enter glycolysis or reduced to sorbitol via aldose reductase to enter the polyol pathway. The poorly diffusible sorbitol is converted via sorbitol dehydrogenase to fructose. Hexokinase, aldose reductase and sorbitol dehydrogenase activities were measured in glomeruli (G) and small arteries (SA) taken from normal and diabetic human kidneys, Hexokinase in diabetic G was 1688, which was significantly decreased from normal, 3147 mmoles/kg-1/h-1. Alodse reductase was significantly elevated in diabetic G,56-6, compared to normal G,10-8 mmoles/kg-1/h-1. In contrast, sorbitol dehydrogenase was significantly depressed in diabetic G, 3-7 VERSUs 10-9 mmoles/kg-1/h-1. The enzymatic changes observed in diabetic G would facilitate accumulation of sorbitol and therefore could contribute to the progression of glomerulosclerosis. The activity of hexokinase was also significantly reduced in SA, whereas aldose reductase and sorbitol dehydrogenase were unchanged.     

Oxidative stress decreases antioxidant enzyme activities in reaggregation cultures of rat brain cells

The brain has been suggested to be especially sensitive to damage by reactive oxygen species. In this study, we examined the effects of hyperoxic conditions on the activities and mRNA levels of antioxidant enzymes in reaggregation cultures of rat forebrain cells. Cultures were exposed to 80% oxygen for 12–60 h starting on Days 17 and 33 in culture. Superoxide dismutase activities and mRNA levels were not affected by hyperoxia, whereas catalase activity was slightly decreased after 24 h in 80% oxygen at Day 17. Glutathione peroxidase activity was markedly decreased already after 12 h of hyperoxia, and decreased activities of glutathione reductase and glucose-6-phosphate dehydrogenase were also noted. The glutathione peroxidase mRNA levels were increased in hyperoxic cultures at Day 17 but not at Day 33. These results suggest that the enzymatic defense mechanisms against reactive oxygen species in the brain are rather weak and deteriorate during oxidative stress but that a potential for compensatory upregulation exists at least during the first postnatal weeks.     

Purification of NAD-dependent mannitol dehydrogenase from celery suspension cultures.

Mannitol dehydrogenase, a mannitol:mannose 1-oxidoreductase, constitutes the first enzymatic step in the catabolism of mannitol in nonphotosynthetic tissues of celery (Apium graveolens L.). Endogenous regulation on the enzyme activity in response to environmental cues is critical in modulating tissue concentration of mannitol, which, importantly, contribute to stress tolerance of celery. The enzyme was purified to homogeneity from celery suspension cultures grown on D-mannitol as the carbon source. Mannitol dehydrogenase was purified 589-fold to a specific activity of 365 mumol h-1 mg-1 protein with a 37% yield of enzyme activity present in the crude extract. A highly efficient and simple purification protocol was developed involving polyethylene glycol fractionation, diethylaminoethyl-anion-exchange chromatography, and NAD-agarose affinity chromatography using NAD gradient elution. Sodium dodecylsulfate gel electrophoresis of the final preparation revealed a single 40-kD protein. The molecular mass of the native protein was determined to be approximately 43 kD, indicating that the enzyme is a monomer. Polyclonal antibodies raised against the enzyme inhibited enzymatic activity of purified mannitol dehydrogenase. Immunoblots of crude protein extracts from mannitol-grown celery cells and sink tissues of celery, celeriac, and parsley subjected to sodium dodecyl sulfate gel electrophoresis showed a single major immuno-reactive 40-kD protein.     

Distribution of Metabolites and Enzymes of Nitrogen Metabolism between the Mesophyll and Paraveinal Mesophyll of Non-Nodulated Glycine max

The distribution of amino acids and key enzymes involved innitrogen metabolism was determined in mesophyll cells (MC),mesophyll protoplasts (MP), and paraveinal mesophyll protoplasts(PVMP) isolated from fully expanded trifoliolate leaves of non-nodulatedsoybean. Qualitative and quantitative differences were foundin the distribution of amino acids, with MP containing the highestconcentrations. Activity of nitrate reductase, glycolate oxidase,glutamine synthetase and glutamate dehydrogenase was measuredin both tissue types and differences in activities between thetissue types were seen. PVMP had high glutamate dehydrogenaseactivity when compared to MP. Activities of glycolate oxidaseand glutamine synthetase were much higher in MP on a protoplastbasis while nitrate reductase activity was similar between thetwo protoplast types. These results, on the distribution ofmetabolites and associated enzymes, are discussed as to theirpossible significance to nitrogen metabolism in the soybeanleaf. Key words: Amino acids, glutamate dehydrogenase, Glycine max, nitrate reductase, nitrogen metabolism, paraveinal mesophyll, protoplasts     

Phospholipase A2 is a differentiation-dependent enzymatic activity for adipogenic cell line and adipocyte precursors in primary culture

Phospholipase A2 enzymatic activity was measured in the teratoma-derived adipogenic cell line 1246 and in adipocyte precursors in primary cultures. It was shown that enzymatic activity was low while the cells were undifferentiated and increased by 20-24-fold after the cells had undergone adipocyte differentiation. The increase of phospholipase A2 activity follows the same time course as that observed for glycerol-3-phosphate dehydrogenase activity used as a marker of differentiation. In contrast, the differentiation-deficient, insulin-independent cell line 1246-3A always contained very low levels of phospholipase A2 activity. Phospholipase A2 activity measured in the 1246 cells was inhibited in a dose-dependent fashion by incubation with ONO-RS-082 and quinacrine which are inhibitors of phospholipase A2 activity. Measurements of arachidonate metabolites in 1246 cells showed that production of prostaglandin F2 alpha by the 1246 cells followed the same time course as the increase of phospholipase A2 activity during differentiation. Similar results were obtained with primary cultures of adipocyte precursors. These results indicate that phospholipase A2 is a differentiation-dependent enzymatic activity for the adipogenic cell line 1246 and for adipocyte precursors in primary culture. These data suggest that metabolic pathways controlled by phospholipase A2 activity could play an important physiological role in adipose tissue differentiation.     

Relative Activities and Characteristics of Some Oxidative Respiratory Enzymes from Conidia of Verticillium albo-atrum

Conidia of Verticillium albo-atrum Reinke and Berthold, collected from shake cultures grown in Czapek broth, were sonified for 4 or 8 minutes or ground frozen in a mortar to obtain cell-free homogenates. These were assayed for certain enzymes associated with respiratory pathways. Malic dehydrogenase was the most active, glucose-6-P and NADH dehydrogenase were less active, NADH-cytochrome c reductase, NADPH dehydrogenase, and cytochrome oxidase were low in activity, and succinic dehydrogenase and succinic cytochrome c reductase were very low to negligible in activity. No NADH oxidase activity was detected.

With the exception of NADH-cytochrome c reductase and possibly succinic dehydrogenase and cytochrome c reductase, there was no evident increase in specific activity of the enzymes during germination. Some NADH-cytochrome c reductase and a small amount of succinic-dehydrogenase and cytochrome c reductase were associated with the particulate fraction from 105,000 × g centrifugation. The other enzymes, including cytochrome oxidase, almost completely remained in the supernatant fraction.

Menadione and vitamin K-S(II) markedly stimulated NADH-cytochrome c reductase activity in the supernatant fraction but had much less effect on NADPH-cytochrome c reductase in this fraction or on either of these enzyme systems in the particulate fraction. Electron transport inhibitors affected particulate NADH- and NADPH-cytochrome c reductase activity but had no effect on these in the supernatant fraction.