Phase I and phase II enzymes produced by Cunninghamella elegans for the metabolism of xenobiotics

Abstract The filamentous fungus Cunninghamella elegans has the ability to metabolize xenobiotics, including polycyclic aromatic hydrocarbons and pharmaceutical drugs, by both phase I and II biotransformations. Cytosolic and microsomal fractions were assayed for activities of cytochrome P450 monooxygenase, aryl sulfotransferase, glutathione S -transferase, UDP-glucuronosyltransferase, UDP-glucosyltransferase, and N -acetyltransferase. The cytosolic preparations contained activities of an aryl sulfotransferase (15.0 nmol min⁻¹ mg⁻¹), UDP-glucosyltransferase (0.27 nmol min⁻¹ mg⁻¹) and glutathione 5-transferase (20.8 nmol min⁻¹ mg⁻¹). In contrast, the microsomal preparations contained cytochrome P450 monooxygenase activities for aromatic hydroxylation (0.15 nmol min⁻¹ mg⁻¹) and N -demethylation (0.17 nmol min⁻¹~' mg⁻¹) of cyclobenzaprine. UDP-glucuronosyltransferase activity was detected in both the cytosol (0.09 nmol min⁻¹ mg⁻¹) and the microsomes (0.13 nmol min⁻¹ mg⁻¹). N -Acetyltransferase was not detected. The results from these experiments provide enzymatic mechanism data to support earlier studies and further indicate that C. elegans has a broad physiological versatility in the metabolism of xenobiotics.     

Vascularization of the lateral muscle of some elasmobranchiomorph fishes

The vascular bed of the locomotor muscle in three shark species and in a holocephalan is investigated and the capillarization quantified by morphometrical methods. The red muscle fibres of Scyliorhinus, Galeus, Etmopterus and Chimaera are well vascularized having respectively 25, 18, 23 and 5 per cent of fibre surface covered by capillaries. The white muscle fibres of Scyliorhinus are far better vascularized than white muscle fibres of Galeus, Etmopterus and Chimaera . The fibre surface covered by capillaries are 9, 1.4, 0.2 and 1.3 per cent, respectively. The intermediate muscle fibres have a vascular supply between that of red and white muscle fibres in all species. If the capillary contact area to the fibres are related to the mitochondrial volume, more similar values are obtained for the vascular supply to all fibres types in all animals. This parameter indicates that there is a definite relation between the capacity of the oxygen transport system and the aerobic metabolic machinery.     

Pyruvate Carboxylase Activity in Primary Cultures of Astrocytes and Neurons

Abstract: The activity of the pyruvate carboxylase was determined in brains of newborn and adult mice as well as primary cultures of astrocytes, of cerebral cortex neurons, and of cerebellar granule cells. The activity was found to be 0.25 ± 0.14, 1.24 ± 0.07, and 1.75 ± 0.13 nmol · min⁻¹· mg⁻¹ protein in, respectively, neonatal brain, adult brain, and astrocytes. Neither of the two types of neurons showed any detectable enzyme activity (i.e., < 0.05 nmol · min⁻¹· mg⁻¹). It is therefore concluded that pyruvate carboxylase is an astrocytic enzyme.     

Copper uptake by free and immobilized cyanobacterium

Abstract Copper uptake in free and immobilized cells of the cyanobacterium Nostoc calcicola has been examined. The immobilized cells invariably maintained a higher profile of Cu intake rate (12.7 nmol mg⁻¹ protein min⁻¹) over the free cells (6.0 nmol mg⁻¹ protein min⁻¹). The total Cu uptake in immobilized cells was almost two and a half-times more than their free cell counterpart under identical experimental conditions. Also, the immobilized cells showed a stronger positive correlation between Cu adsorption and uptake. The results have been discussed in terms of improved metabolic efficiency of immobilized cells.     

PHOSPHATE ACTIVATED GLUTAMINASE ACTIVITY AND GLUTAMINE UPTAKE IN PRIMARY CULTURES OF ASTROCYTES

Abstract— Uptake and release of glutamine were measured in primary cultures of astrocytes together with the activity of the phosphate activated glutaminase (EC 3.5.1.2). In contrast to previous findings of an effective, high affinity uptake of other amino acids (e.g. glutamate, GABA) no such uptake of glutamine was observed, though a saturable, concentrative uptake mechanism did exist (K _m = 3.3 ± 0.5 m m ; V _max= 50.2 ± 12.6 nmol ± min⁻¹± mg⁻¹). The phosphate activated glutaminase activity in the astrocytes (6.9 ± 0.9 nmol ± min⁻¹± mg⁻¹) was similar to the activity found in whole brain (5.4 ± 0.7 nmol ± min ^−l± mg⁻¹), which may contrast with previous findings of a higher activity of the glutamine synthetase (EC 6.3.1.2) in astrocytes than in whole brain. The observations are compatible with the hypothesis of an in vivo flow of glutamate (and GABA) from neurons to astrocytes where it is taken up and metabolized, and a compensatory flow of glutamine towards neurons and away from astrocytes although the latter cell type may be more deeply involved in glutamine metabolism than envisaged in the hypothesis.     

UPTAKE AND METABOLISM OF GLUTAMATE IN ASTROCYTES CULTURED FROM DISSOCIATED MOUSE BRAIN HEMISPHERES

Abstract— Uptake kinetics of l -glutamate in cultured, normal glia cells obtained from the brain hemispheres of newborn mice were measured together with the activities of the glutamate metabolizing enzymes, glutamic-oxaloacetate-transaminase, glutamate dehydrogenase and glutamine synthetase. During 3 weeks of culturing, the activities of the enzymes rose from low neonatal values toward the levels in the adult brain (206, 12.3 and 25.9 nmol. min⁻¹. mg⁻¹ cell protein for the three enzymes, respectively). The uptake kinetics indicated an unsaturable component together with an uptake following Michaelis-Menten kinetics with a K_m of 220 μ m and a V _max of 7.9 nmol. min⁻¹. mg⁻¹ cell protein. The saturable glutamate uptake was inhibited by d -glutamate, l -aspartate and α-aminoadipate whereas l -glutamine, GABA and glutarate had no effect. The uptake which was Ca²⁺-independent had a K_m for sodium of 18m m and it was stimulated by an increase in the external potassium concentration from 5 to 10 and 25 m m. The results suggest that glia cells are important for the uptake of glutamate from synaptic clefts and for the subsequent metabolism of glutamate.     

Characterization of Neuronal Amino Acid Transporters: Uptake of Nitric Oxide Synthase Inhibitors and Implication for Their Biological Effects

Abstract: In the present study we investigated uptake of the nitric oxide (NO) synthase inhibitors N ^G-methyl- l -arginine and N ^G-nitro- l -arginine by the mouse neuroblastoma × rat glioma hybrid cell line NG108-15. Uptake of N ^G-methyl- l -arginine was characterized by biphasic kinetics ( K _m1 = 8 µmol/L, V _max1 = 0.09 nmol × mg⁻¹× min⁻¹; K _m2 = 229 µmol/L, V _max2 = 2.9 nmol × mg⁻¹× min⁻¹) and was inhibited by basic but not by neutral amino acids. Uptake of N ^G-nitro- l -arginine followed Michaelis-Menten kinetics ( K _m = 265 µmol/L, V _max = 12.8 ± 0.86 nmol × mg⁻¹× min⁻¹) and was selectively inhibited by aromatic and branched chain amino acids. Further characterization of the transport systems revealed that uptake of N ^G-methyl- l -arginine is mediated by system y⁺, whereas systems L and T account for the transport of N ^G-nitro- l -arginine. In agreement with these data on uptake of the inhibitors, l -lysine and l -ornithine antagonized the inhibitory effects of N ^G-methyl- l -arginine on bradykinin-induced intracellular cyclic GMP accumulation, whereas l -tryptophan, l -phenylalanine, and l -leucine interfered with the effects of N ^G-nitro- l -arginine. These data suggest that rates of uptake are limiting for the biological effects of NO synthase inhibitors.     

Comparative estimations of the energy content of fish tissue from bomb calorimetry, wet oxidation and proximate analysis

SUMMARY. Four methods were used to determine the energy content of somatic tissues of Perca fluviatilis . Two forms of direct calorimetry (both adiabatic and non-adiabatic) and wet (dichromate) oxidation gave similar results. When energy contents were calculated from proximate analysis using accepted conversion factors (9.45 cal mg⁻¹ for lipid, 5.65 cal mg⁻¹ for protein) results were higher than those from the other methods. The discrepancy was eliminated when a lower, directly determined energy content for the extracted lipid fraction was used.
Some historic and technical aspects of the comparison are discussed.     

Alternative oxidase from Arum and soybean: Its stabilization during purification

Complete purification of the alternative oxidase from plant mitochondria has not been achieved successfully, because of its instability on solubilization. We report here that the addition of pyruvate to the isolation medium stabilizes the activity of the solubilized enzyme. A procedure is described for the rapid isolation and partial purification of the cyanide-insensitive alternative oxidase from both Arum maculatum and soybean cotyledon ( Glycine max ) mitochondria. The degree of purification was 16- and 74-fold for Arum and soybean enzyme, respectively. The specific activities increased from 1 300 to 20 300 nmol oxygen consumed mg⁻¹ protein min⁻¹ (using duroquinol as substrate) after purification for the Arum erizyme and from 6 to 445 nmol oxygen consumed mg⁻¹ protein min⁻¹ for the soybean enzyme. A turnover for the partially purified Arum enzyme was estimated to be 47 electrons s⁻¹.
The partially purified enzyme from both Arum and soybean cotyledon mitochondria was sensitive to alternative oxidase inhibitors such as salicylhydroxamic acid, n -propyl gallate and octyl gallate, but not to myxottriazol, KCN or antimycin A. The activity of the enzyme could be stimulated by pyruvate, but not by malate and suceinate. The stability of the purified enzyme was also dependent on the continued presence of pyruvate. In the absence of pyruvace, the enzyme activity was lost in a time-dependent manner and the ability of pyruvate to recover the activity was also irreversibly lost.     

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

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

N2-dependent growth and nitrogenase activity in the metal-metabolizing bacteria, Geobacter and Magnetospirillum species

Cells of Geobacter metallireducens , Magnetospirillum strain AMB-1, Magnetospirillum magnetotacticum and Magnetospirillum gryphiswaldense showed N₂-dependent growth, the first anaerobically with Fe(III) as the electron acceptor, and the latter three species microaerobically in semi-solid oxygen gradient cultures. Cells of the Magnetospirillum species grown with N₂ under microaerobic conditions were magnetotactic and therefore produced magnetosomes. Cells of Geobacter metallireducens reduced acetylene to ethylene (11.5 ± 5.9 nmol C₂H₄ produced min⁻¹ mg⁻¹ cell protein) while growing with Fe(III) as the electron acceptor in anaerobic growth medium lacking a fixed nitrogen source. Cells of the Magnetospirillum species, grown in a semi-solid oxygen gradient medium, also reduced acetylene at comparable rates. Uncut chromosomal and fragments from endonuclease-digested chromosomal DNA from these species, as well as Geobacter sulphurreducens organisms, hybridized with a nifHDK probe from Rhodospirillum rubrum , indicating the presence of these nitrogenase structural genes in these organisms. The evidence presented here shows that members of the metal-metabolizing genera, Geobacter and Magnetospirillum , fix atmospheric dinitrogen.     

Growth, incidence and activities of dissimilatory sulfate-reducing bacteria in the human oral cavity

Abstract Viable counts and activities of sulfate-reducing bacteria were determined in the oral cavities of 12 healthy volunteers. Of these, 10 harboured viable sulfate-reducing bacteria populations. Six separate sites were sampled: the posterior tongue, anterior tongue, mid buccal mucosa, vestibular mucosa, supragingival plaque and subgingival plaque. Sulfate-reducing bacteria occurred in all areas, with the highest incidence in supragingival plaque. Viable counts and sulfate-reducing activities in each of the regions varied from 0 to 10⁸ cfu (g wet weight)⁻¹ and from 0 to 50 nmol (g wet weight) ⁻¹ h⁻¹, respectively. As sulfate-reducing bacteria can be detected in the oral cavity, they may potentially be involved in terminal oxidative processes carried out by the microflora of the mouth.     

Mycorrhiza formation on Norway spruce (Picea abies) roots affects the pathway of anaplerotic CO2 fixation

Activities of carboxylation enzymes were analyzed in the mycelium of the mycorrhizal fungus Amanita muscaria (L. ex Fr.) Hooker, in non-mycorrhizal short roots of Norway spruce ( Picea abies [L.] Karst.) and in myconhizas of these two partners. While pyruvale carboxylase (PC, EC 6.4.1.1) and phosphoenolpyruvate carboxykinase activities (PEPCK.EC 4.1.1.49) could be detected in the mycelium of A. muscaria , phosphoenolpyruvate carboxyknase (PEPC, EC 4.1.1.31) was only active in root tissue. In A. muscaria , PC activity was generally low (around 10 nmol mg^−tprotein min⁻) but PEPCK activity was above 250 nmol mg⁻¹ protein min⁻¹. Mycorrhizal development on short roots decreased PEPC activity by more than 75%, although dilution by the fungal biomass in mycorrhizas was only 35%. This reduction in activity was paralleled by a decreased content of PEPC protein. By means of micro-analytical methods it was shown that PEPC activity was lowest in the central zones of the mycorrhizas, Whereas PEPC activity was highest in the corresponding central sections in non-mycorrhizal short roots. ¹⁴CO₂ labelling, on the other hand, revealed that in vivo CO₂ fixation was higher in mycorrhizas compared to non-mycorrhizal short roots. It is concluded that fungal carboxylases (probably PEPCK) are important for anaplerotic CO₂ fixation during nitrogen assimilation in mycorrhizas of Norway spruce.     

Uptake of glutamine and glutamate by the dinitrogen-fixing cyanobacterium Anabaena sp. PCC7120

Abstract Whole cells of the dinitrogen-fixing cyanobacterium Anabaena sp. PCC7120 exhibited K _m values for l -glutamine and l -glutamate of 33 μM and 0.5 mM, respectively. V _max of uptake was ca. 30 nmol mg⁻¹ (chlorophyll) min⁻¹ for both amino acids. The similar pattern of sensitivity to other amino acids exhibited by both transport activities suggests that a common transport system is involved in glutamine and glutamate uptake by this cyanobacterium.     

Content of low-molecular-weight thiols during the imbibition of Pea seeds

The metabolism of low-molecular-weight thiols was investigated in seeds of Pisum sativum L. cv. Kleine Rheinländerin during imbibition in water for 14 h. The amount of oxidized glutathione (GSSG) decreased from 319 nmol (g dry weight)⁻¹ in dry seeds to 38 nmol (g dry weight)⁻¹ within the first 14 h of imbibition. The decrease may have been due to the reduction of GSSG to reduced glutathione (GSH), catalyzed by the enzyme glutathione reductase (GR; EC 1.6.4.2). The enzyme activity was high in dry seeds [25 nkat (g dry weight)⁻¹] and decreased to 20 nkat (g dry weight)⁻¹ within 14 h of imbibition. The activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) decreased from 100 nkat (g dry weight)⁻¹ in dry seeds to 67 nkat (g dry weight)⁻¹ after 14 h of imbibition. Within 14 h the amount of γ-glutamyl-cysteine (γ-GC) decreased from 135 to 38 nmol (g dry weight)⁻¹, whereas the cysteine content rose from 81 nmol (g dry weight)⁻¹ in dry seeds to a maximum of 170 nmol (g dry weight)⁻¹ after 12 h of imbibition, which may be due to the degradation of γ-GC into cysteine.     

Cholinergic constituents in plants: Characterization and distribution of acetylcholine and choline

Acetylcholine in plants was identified by gas chromatography/mass spectrometry. Acetylcholine was found in the following species from 13 families: Betula pendula, Codiaeum variegatum, Ilex opaca, Liquidambar styraciflua, Lonicera japonica, Phaseolus aureus, Phaseolus vulgaris, Pisum sativum, Plantago rugelli, Populus grandidentata, Prunus serotina, Rhus copallina, Smilax hispida, Viburnum dilatatum , and Zea mays . Levels of acetylcholine in leaves ranged from a low of 0.14 ± 0.05 (mean ± SEM) nmol (g fresh weight)⁻¹ in I. opaca to a high of 53 ± 6.6 nmol (g fresh weight)⁻¹ in P. aureus . Acetylcholine was found in all tissues examined regardless of the organ (leaves, stems, or roots) or developmental stage (seedlings, mature plants, or seeds). For P. aureus , continuous light exposure increased acetylcholine levels of leaves, and decreased levels in stem when compared to dark controls. Levels of choline, a precursor of acetylcholine, found in leaves ranged from a low of 84 ± 7.0 nmol (g fresh weight)⁻¹ in L. styraciflua to a high of 3700 ± 200 nmol (g fresh weight)⁻¹ in P. aureus . With these findings, three out of the four components of the cholinergic system have now been identified in plants.     

Carbon monoxide dehydrogenase and acetate thiokinase in Methanothrix soehngenii

Abstract In Methanothrix soehngenii acetate is first activated by an acetate thiokinase rather than a phosphotransacetylase. The specific activity of the acetate thiokinase was 5.29 μmol acetate activated min⁻¹ mg⁻¹ protein with a half maximum rate at 0.74 mM acetate and at 0.047 mM CoA. In cell-free extracts a CO-dehydrogenase activity was measured of 3.02 μmol min⁻¹ mg⁻¹ protein with a half maximum rate at 0.44 mM CO and at 0.18 mM methylviologen. NADP and NAD could not replace methylviologen. F₄₂₀ showed only low activity as electron acceptor.     

Peroxidase isoenzymes in the defense response of Capsicum annuum to Phytophthora capsici

Quantitative and qualitative changes in isoperoxidase patterns from stems of three cultivars of pepper ( Capsicum annuum L.). one susceptible, one intermediate and one resistant, were found upon inoculation with Phytophthora capsici using a decapitation method. The peroxidase activity was determined in the intercellular fluid as well as in the cytosolic fraction of the necrotic, healthy and intermediate zones of stems of the three cultivars, 6 days after inoculation. In the intercellular fluid, peroxidase activity of the susceptible cv. Yolo Wonder increased somewhat from 4.7 (healthy zone) to 12.9 (intermediate zone) μmol mg⁻¹ protein min⁻¹, whereas in the intermediate cv. Americano, the peroxidase activity decreased from 123 (healthy zone) to 78 (intermediate zone) μmol mg⁻¹ protein min⁻¹. The most dramatic increase (5.7 to 662 μmol mg⁻¹ protein min⁻¹) in intercellular peroxidase activity was found in the resistant cv. Smith-5. This, in conjunction with the appearance of an additional acidic isoperoxidase (pI 4.4) specific for the cv. Smith-5, could be the reason for the resistance of this cultivar against the fungus attack. The release of peroxidase into the intercellular space as a defense reaction was confirmed by histochemical analysis, showing that peroxidase activity occurred in the intercellular spaces of those stems of the resistant cultivar that had not yet been invaded by the fungus, but was detected neither in the other cultivars nor in the intercellular spaces of such stems of the intermediate and susceptible cultivars that contained growing mycelium of P. capsici. The lack of staining in the intercellular spaces of the susceptible cultivars could be attributed to their low content in peroxidase.     

Oxygen consumption in Oreochromis niloticus (L.) in relation to development, salinity, temperature and time of day

Oxygen consumption of Oreochromis niloticus at different stages of development was studied in relation to salinity, temperature and time of day, using a Warburg apparatus. The oxygen consumption of newly hatched (0–14 h) larvae was 3.40 μl O₂ larva⁻¹ h⁻¹, of older yolk sac larvae 10.09 μl O₂ larva⁻¹ h⁻¹, and of one-month-old fry 32.99 μl O₂ larva⁻¹ h⁻¹. The QO₂ values showed a decrease with development and growth, ranging from 21.2–26.0 μl O₂ mg⁻¹ h⁻¹ in newly hatched larvae to 2.97 μl mg⁻¹ h⁻¹ in one-month-old fry. Changes in oxygen consumption occurred with salinity, the highest being at 17%o. Active larvae (12-24 mm T.L.) showed a doubling of consumption with a 10° C rise in temperature, and their Q₁₀ factor increased from 2.25 to 3.43 with increasing size. Day-old yolk-sac larvae, late yolk-sac larvae (5 days old) and fry of 12 14 mm length all showed a depression in oxygen consumption at midnight followed by a dawn rise.     

Induction of nitrate reductase in needles of Scots pine seedlings by NOX and NO3−

The possibility to induce nitrate reductase (NR; EC 1.6.6.2) in needles of Scots pine ( Pinus sylvestris L.) seedlings was studied. The NR activity was measured by an in vivo assay. Although increased NR activities were found in the roots after application of NO₃⁻, no such increase could be detected in the needles. Detached seedlings placed in NO₃⁻ solution showed increasing NR activities with increasing NO₃⁻ concentrations. Exposure of seedlings to NO_x (70–80 ppb NO₂ and 8–12ppb NO) resulted in an increase of the NR activity from 10–20 nmol NO₂⁻ (g fresh weight)⁻¹ h⁻¹ to about 400 nmol NO₂⁻ (g fresh weight)⁻¹ h⁻¹. This level was reached after 2–4 days of exposure, thereafter the NR activity decreased to about 200 nmol NO₂⁻ (g fresh weight)⁻¹ h⁻¹. Analyses of free amino acids showed low concentrations of arginine and glutamine in NO_x-fumigated seedlings compared to corresponding controls.