Characterization of catecholamine uptake2 in isolated cardiac myocytes

Extraneuronal catecholamine uptake was investigated in isolated quiescent rat myocardial cells. By administration of (³H-)(–)noradrenaline concentration of 22 nmol/l up to 1000 mol/l the following data were obtained: (1) The K_M of the uptake process amounted to 260 mol/l, the V_max to 4.24 nmol/(10 min × mg Protein) corresponding to 179 nmol/(min × g_WWt)(WWT = Wet Weight). (2) The uptake was largely inhibited by the uptake₂-inhibitors corticosterone (100 mol/l), isoprenaline (IC so = 30.6 mol/l), and O-methylisoprenaline (IC₅₀ = 2.1 pmol/l), but not by the uptake₁-inhibitors cocaine (100 mol/l) and desipramine (10 mol/l). (3) The affinity-values K_M and IC₅₀ closely agreed with those already known, but the V_max-value was higher than those obtained in whole rat hearts by a factor of at least 1.79. This is caused presumably by the voltage dependence of the uptake mechanism and the resulting inhibition of uptake 2 during the periods of depolarisation in beating hearts of other studies.     

Reciprocal Innervation between Serotonergic and GABAergic Neurons in Raphe Nuclei of the Rat

Midbrain slices containing the dorsal and medial raphe nuclei were prepared from rat brain in order to study serotonergic-GABAergic interaction. The slices were loaded with either [³H] serotonin or [³H]GABA, superfused and the electrically induced efflux of radioactivity was determined. The GABA_A receptor agonist muscimol (3 to 30 M) and the GABA_B receptor agonist baclofen (30 and 100 M) inhibited [³H]serotonin and [³H]GABA release. These effects of muscimol were reversed by the GABA_A antagonists bicuculline (100 M). The GABA_B antagonist phaclofen (100 M) also antagonized the baclofen-induced inhibition of [³H]serotonin and [³H]GABA release. Phaclofen by itself increased [³H]serotonin release but it did not alter [³H]GABA overflow. Muscimol (10 M) and baclofen (100 M) also inhibited [³H]serotonin release after depletion of GABAergic neurons by isoniazid pretreatment. These findings indicate the presence of postsynaptic GABA_A and GABA_B receptors located on serotonergic neurons. The 5-HT_1A receptor agonist 8-OH-DPAT (0.01 to 1 M) and the 5-HT_1B receptor agonist CGS-12066A (0.01 to 1 M) inhibited the electrically stimulated [³H]serotonin and [³H]GABA release. The 5-HT_1A antagonist WAY-100135 (1 M) was without effect on [³H]serotonin and [³H]GABA efflux by itself but it reversed the 8-OH-DPAT-induced transmitter release inhibition. During KCl (22 mM)-induced depolarization, tetrodotoxin (1 M) did not alter the inhibitory effect of CGS-12066A (1 M) on [³H]GABA release, it did blocked, however, the ability of 8-OH-DPAT (1 M) to reduce [³H]GABA efflux. After depletion of raphe serotonin neurons by p-chlorophenylalanine pretreatment, CGS-12066A (1 M) still inhibited [³H]GABA release whereas in serotonin-depleted slices, 8-OH-DPAT (1 M) was without effect on the release. We conclude that reciprocal influence exists between serotonergic projection neurons and the GABAergic interneurons or afferents in the raphe nuclei and these interactions may be mediated by 5-HT_1A/B and GABA_A/B receptors. Both synaptic and non-synaptic neurotransmission may be operative in the 5-HTergic-GABAergic reciprocal interaction which may serve as a local tuning in the neural connection between cerebral cortex and midbrain raphe nuclei.     

Role of mitochondria in ethanol tolerance of Saccharomyces cerevisiae

The presence of active mitochondria and oxidative metabolism is shown to be essential to maintain low inhibition levels by ethanol of the growth rate (), fermentation rate (v) or respiration rate () of Saccharomyces cerevisiae wild type strain S288C. Cells which have respiratory metabolism show K _i (ethanol inhibition constant) values for , v and , higher (K _i>1 M) than those of petite mutants or grande strains grown in anaerobiosis (K _i=0.7 M). In addition, the relationship between or v and ethanol concentration is linear in cells with respiratory metabolism and exponential in cells lacking respiration. When functional mitochondria are transferred to petite mutants, the resulting strain shows K _i values similar to those of the grande strain and the inhibition of and v by increasing ethanol concentrations becomes linear.     

Effects of <Emphasis Type="Italic">in vitro</Emphasis>rooting environments and irradiance on growth and photosynthesis of strawberry plantlets during acclimatization

Strawberry (Fragaria ananassaDuch. cv. Fengxiang) plantlets were cultured under two in vitroenvironments for rooting, and then acclimatized under two ex vitroirradiance conditions. At the end of rooting stage plant height, fresh weight and specific leaf area of T1-plants grown under high sucrose concentration (3 sucrose), low photosynthetic photon flux density (30 mol m^–2 s^–1) and normal CO₂ concentration (350–400 l l^–1) were significantly higher than those of T2-plantlets grown under low sucrose concentration (0.5), high photosynthetic photon flux density (90 mol m^–2 s^–1) and elevated CO₂ concentration (700–800 l l^–1). But T2-plantlets had higher net photosynthetic rate (Pn), effective photochemical quantum yield of PSII (_PSII), effective photosynthetic electron transport rate (ETR), photochemical quenching (q_P) and ratio of chlorophyll fluorescence yield decrease (Rfd). After transfer, higher irradiance obviously promoted the growth of plantlets and was beneficial for the development of photosynthetic functions during acclimatization. T2-plantlets had higher fresh weight, leaf area, _PSII and ETR under higher ex vitroirradiance condition.     

Somatic embryogenesis in callus cultures of Rosa hybrida L. cv. Landora

Callus was initiated from immature leaf and stem segments of rose (Rosa hybrida cv. Landora) and subcultured every four weeks on a basal medium of half-strength Murashige & Skoog (1962) salts plus 30 g l^-1 sucrose (1/2 MS) and supplemented with 2.2 M BA, 5.4 M NAA and 2.2–9.0 M 2,4-D. Embryogenic callus and subsequently somatic embryos were obtained from 8-week-old callus culture on 1/2 MS+2.2 M BA+0.05 M NAA+0.3 M GA₃+200–800 mg l^-1 L-proline. Long-term cultures were established and maintained for up to 16 months by repeated subculture of embryogenic callus on L-proline deficient medium. About 12% of cotyledonary stage embryos taken from cultures cold-stored at 8±1°C for 4 days germinated on 1/2 MS+2.2 M BA+0.3 M GA₃+24.7 M adenine sulphate.Abbreviations BA benzyladenine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid     

Ecotoxicity of lead to the zebra musselDreissena Polymorpha,Pallas

The effect of lead on the filtration rate of the zebra musselDreissena polymorpha was investigated, together with the accumulation of Pb in the soft tissues of the mussels. The NOEC-filtration was 116 g.l^–1 (0,56 mol.l^–1) and the EC₅₀-filtration was 370 g.l^–1 (1.79 mol.l^–1). The NOEC-accumulation was the concentration found in the control water (1.4g.l^–1). These experiments show that the EC₅₀-filtration for Pb is similar to that for Cd, higher than that for Cu and lower than that for Zn. The water quality criteria for lead allow 25 g Pb.l^–1 in surface water. This will not cause short-term effects. Long-term effects may, however, occur, since an accumulation of Pb as low as 16 g.l^–1 was recorded in this study.     

Development of high frequency multiple shoot formation in Persian clover (<Emphasis Type="Italic">Trifolium resupinatum</Emphasis> L.)

An efficient and reliable micropropagation system for Persian clover (Trifolium resupinatum L.) was developed using different explants and media. Node, hypocotyl and cotyledonary node explants were cultured on Murashige and Skoog (MS) medium supplemented with combinations of either 6-benzyladenine (BA) and indole-3-butyric acid (IBA) or BA, Kinetin (KIN) and IBA. Direct multiple shoots developed within 6weeks in all explants in most media tested. The best shoot multiplication capacity was obtained from cotyledonary node explants on MS medium containing 7.1M BA and 1M IBA or 14.1M BA and 1M IBA. Elongated shoots were rooted on either MS medium alone or combination with different concentrations of indole-3-butyric acid (IBA), indole-3-acetic acid (IAA) and -naphthaleneacetic acid (NAA). High rooting was achieved in half strength MS medium containing 8M IBA.     

In vitro culture of Aloe Barbadensis Mill.: Micropropagation from vegetative meristems

A method for rapid and highly effective plant micropropagation from vegetative meristems was established for Aloe barbadensis Mill. Plant micropropagation was achieved culturing apices on medium containing 1.1 M 2,4-dichlorophenoxyacetic acid and 2.3 M kinetin for 15–30 days. High morphogenetic ability was maintained by transferring explants (after 60 days) on media containing 0.11 M 2,4-dichlorophenoxyacetic acid and 2.2 M 6-benzylaminopurine.     

Stereospecificity and electrogenicity of amino acid transport in Riccia fluitans

In the aquatic liverwort Riccia fluitans, membrane depolarization (_m), change in membrane conductance (g_m), and current-voltage (I-V) characteristics in the presence of different amino acids as well as the uptake of ¹⁴C-labeled amino acids were measured. L-isomers of the tested amino acids generate larger electrical effects (_m, g_m) than D-isomers, and the I-V characteristics show that the positive electrical inward-current of 20 mA m^-2 generated by 0.5 mM D-serine is only about 50% of the current generated by adding 0.5 mM L-serine. Whereas - and -amino acids rapidly depolarize the membrane to the same extend, with -aminobutyric acid (-AB) and dipeptides no significant electrical effects have been measured. The uptake kinetics of ¹⁴C-labeled amino acids display three components: (I) A saturable high-affinity component with K_s-values of 48 M D-alanine, 12 M -aminoisobutyric acid (AIB), 9 M L-alanine, 8 M L-proline, and 6 M L-serine, respectively; (2) an apparently linear low-affinity component, and (3) an also linear but unspecific component at concentrations >20 times the given K_s-value. Uptake of ¹⁴C-labeled AIB can be inhibited competitively by all tested neutral amino acids, the L-isomers being more effective than the D-isomers, as well as by ammonium or methylamine. Vice versa, AIB competitively inhibits uptake of L-serine and L-alanine. It is concluded that an uncharged stereospecific carrier for the investigated amino acids exists in the plasmalemma of Riccia fluitans. Accumulation ratios of about 50 suggest secondary active transport driven by a transmembrane electro-chemical gradient (mainly _m) which is generated by the electrogenic proton pump. It is suggested that this carrier binds to the amino group forming either a charged binary complex with positively charged amines (Felle 1980), or an uncharged complex with -AB or dipeptides, whereas electrogenic transport of - and -amino acids is mediated by a ternary carrier complex, probably charged by a proton.Symbols and Abbreviations _m membrane potential (mV) - E_co equilibrium potential (mV) of the transport system - g_m membrane (slope) conductance (Sm^-2) - g_m change in g_m - I-V curve current-voltage curve - AIB -aminoisobutytric acid - -AB -aminobutyric acid     

The Synthesis and Antiviral Activity of Glycyrrhizic Acid Conjugates with α-D-Glucosamine and Some Glycosylamines

Glycyrrhizic acid and its 30-methyl ester were conjugated with 2-amino-1,3,4,6-tetra-O-acetyl-2-deoxy--D-glucopyranose, 2,3,4,6-tetra-O-acetyl--D-glucopyranosyl amine, 2,3,4-tri-O-acetyl--L-arabinopyranosyl amine, 2-acetamido-2-deoxy--D-glucopyranosyl amine, and -D-galactopyranosyl amine using N,N-dicyclohexylcarbodiimide and its mixtures with N-hydroxybenzotriazole. Structures of the conjugates were confirmed by IR, UV, ¹H, and ¹³C NMR spectroscopy. The glycoconjugate with the residues of 2-acetamido-2-deoxy--D-glucopyranosyl amine in the carbohydrate part of its molecule exhibited antiviral activity (ID₅₀ 4 g/ml) toward the herpes simplex type 1 virus (HSV-1) in the VERO cell culture. Two compounds demonstrated anti-HIV-1 activity (50–70% inhibition of p24) in a culture of MT-4 cells at concentrations of 0.5–20 g/ml.     

Sisal plant regeneration via organogenesis

Callus was initiated from in vitro grown immature leaf and ex vitro grown mature leaf and rhizome explants of Agave sisalana Perr. ex. Engelm, on MS medium containing 2,4-D (9.05 M) and kinetin (4.6 M) or 2,4-D (9.05 M), kinetin (4.6 M) and CH (1000 mg l^–1) or mod. MS (NH₄NO₃, 1500 mg l^–1) containing 2,4-D (9.05 M) and kinetin (4.6 M). Light was essential for callus formation which, however, was different in three types of explants on three different media compositions. Increasing NH₄ ⁺had a negative impact while addition of CH had a positive impact on callus formation. Shoot regeneration from callus from CH-supplemented medium only was achieved for rhizome and immature leaf tissues. The highest rate of regeneration was obtained with BA (26.6 M) as the sole hormone. Shoot buds g^–1 callus varied according to BA concentrations. Shoot proliferation rate increased on half-strength MS medium containing BA (8.9 M). Microshoots developed on MS medium containing BA (2.22 M) and GA₃ (1.44 M) and finally rooted on MS medium containing IAA (11.42 M). Acclimatized rooted plantlets are growing satisfactorily in ex vitro. This is the first report on plant regeneration via organogenesis of A. sisalana.     

The induction and repression of benzene and catechol oxidizing capacity of Pseudomonas putida ML2 studied in perturbed chemostat culture

The oxidation of catechol, an intermediate in benzene catabolism, was studied using transient variations in dissolved oxygen tension (DOT) when a succinate limited steady state culture of Pseudomonas putida ML2 was perturbed with a pulse of another substrate. A model was developed and tested for the effect of fluctuations in oxidizing enzyme activity on DOT. It was found that the rate of induction of catechol oxidizing enzymes was independent of dilution rate up to a relative growth rate /_max of 0.75. Only at higher dilution rates was catabolite repression observed.Abbreviations DOT dissolved oxygen tension - K _L a gas transfer coefficient - specific growth rate - _max maximum specific growth rate - K_s substrate saturation constant     

Promotion of flowering in apple trees with gibberellin A4 and C-3 epi-gibberellin A4

The proportion of spurs flowering on apple trees (Malus domestica Borkh. cv Golden Delicious) displaying a high degree of alternate-year flowering was increased in the off year by gibberellin A₄ (GA₄) and C-3 epi-GA₄ applied in the previous year. When applied 4.5 weeks after anthesis amounts of GA₄ ranging from 3 to 300 g per spur and 25 or 50 g of C-3 epi-GA₄ per spur were effective. Treatments with GA₄ made seven weeks after anthesis were less effective. A combination of 30 g GA₄ and 30 g zeatin (6-(4-hydroxy-3-methylbut-trans-2-enylamino)purine) promoted flowering at both treatment times, and tended to be more effective than GA₄ alone.Abbreviation GA gibberellin or gibberellin-like substance Contribution No. 618     

The effects of aflatoxin B1 on immature germinating maize (Zea mays) embryos

Immature maize (Zea mays L.) embryos were treated with aflatoxin B₁ concentrations, ranging from 0.1 g ml^–1 to 25 g ml^–1. Below 5 g ml^–1 aflatoxin B₁, root and shoot elongation was not significantly inhibited. Ultrastructurally, root tip cells showed little deterioration, except a possible diffused clearing in mitochondria and plastids. As the toxin concentration was increased above 5 gml^–1, shoot, and particularly root elongation, was progressively inhibited. Associated with this, there was an apparent decrease in the ribosome population. Furthermore, membranes, particularly the vacuolar membrane, became abnormal and vacuolar distension occurred. At 20 and 25 g ml^–1, these effects were exacerbated, and mitochondria and plastid structure was disrupted. At these concentrations, there was evidence of a disruption in lipid metabolism. The results are discussed in the context of known aflatoxin effects on cellular control mechanisms and ultrastructure in animal systems.     

Transient state characteristics of adaptation to changes in light conditions for the cyanobacterium Oscillatoria agardhii

Transitions in growth irradiance level from 92 to 7 Em^-2 s^-1 and vice versa caused changes in the pigment contents and photosynthesis of Oscillatoria agardhii. The changes in chlorophyll a and C-phycocyanin contents during the transition from high to low irradiance (HL) were reflected in photosynthetic parameters. In the LH transition light utilization efficiencies of the cells changed faster than pigment contents. This appeared to be related to the lowering of light utilization efficiencies of photosynthesis. As a possible explanation it was hypothesized that excess photosynthate production led to feed back inhibition of photosynthesis. Time-scales of changes in the maximal rate of O₂ evolution were discussed as changes in the number of reaction centers of photosystem II in relation to photosynthetic electron transport. Parameters that were subject to change during irradiance transitions obeyed first order kinetics, but hysteresis occurred when comparing HL with LH transients. Interpretation of first order kinetic analysis was discussed in terms of adaptive response vs changes in growth rate.Non-standard abbreviations Chla chlorophyll a - CPC C-phycocyanin - PS II photosystem II - PS I photosystem I - RC II reaction center of photosystem II - P photosynthetic O₂-evolution - I irradiance, Em^-2 s^-1 - light utilization efficiency of cells, mmol O₂·mg dry wt^-1·h^-1/Em^-2 s^-1 - light utilization efficiency of photosynthetic apparatus, mol O₂·mol Chla ^-1·h^-1/Em^-2 s^-1 - P_max maximal rate of O₂ evolution by cells, mol O₂·mg dry wt^-1·h^-1 - P_max maximal rate of O₂ evolution by photosynthetic apparatus, mol O₂·mol·Chla ^-1·h^-1 - LL low light, E m^-2 s^-1 - HL high light, E m^-2 s^-1 - LH low to high light transition - HL high to low light transition - k specific rate of adaptation, h^-1 - specific growth rate, h^-1 - Q pool size of cell constituent, mol·mg dry wt^-1 - q net synthesis rate of cell constituent, mol·mg dry wt^-1·h^-1     

Electron transfer from cytochrome f to photosystem I in green algae

The time course of P700⁺ reduction and cytochrome f oxidation following a single-turnover flash excitation of photosystem I was measured under various conditions in different strains of green algae. P700⁺ was reduced with a half-time of 4 s. The rate of cytochrome f oxidation was found to depend widely on physiological factors. Reversible transitions are described from a slow-oxidation state (t _1/2=500 s) to a fast-oxidation state (t _1/2=80 s). The addition of ionophore strongly favours and stabilizes the fast-oxidation state. We suggest that these transitions reflect either reversible association between the cytochrome bf complex and the reaction center of photosystem I or changes in the mobility of oxidized plastocyanin. The transitions might be under the control of the membrane potential or the intracellular ATP content. The relation of these reversible transitions with the light state transitions, and their possible involvement in a switch from linear to cyclic electron transfer, are discussed.Abbreviations cyt cytochrome - DCHC dicyclohexyl-18-crown-6 - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DNP-INT dinitrophenylether of iodonitrothymol - FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - LHC light harvesting complex - PC plastocyanin - PS I photosystem I     

Changes of photosynthetic parameters in cucumber leaves under Cu, Cd, and Pb stress

The effects of Cu, Cd, and Pb toxicity on photosynthesis in cucumber leaves (Cucumis sativus L.) were studied by the measurements of gas exchange characteristics, chlorophyll (Chl) fluorescence parameters, and Chl content. Concentrations of metals in sequence of 20 M Cu, 20 and 50 M Cd, and 1 000 M Pb decreased the plant dry mass to 50–60 % after 10 d of treatment whereas 50 M of Cu decreased it to 30 %. The content of Cd in leaves of plants treated with 50 M Cd was three times higher than the contents of Cu and Pb after plant treatment with 50 M Cu or 1 000 M Pb. Hence Cd was transported to leaves much better than Cu and Pb. Nevertheless, the net photosynthetic rate and stomatal conductance in leaves treated with 50 M Cu or Cd were similarly reduced. Thus, Cu was more toxic than Cd and Pb for photosynthesis in cucumber leaves. None of the investigated metals decreased internal CO₂ concentrations. Also the effect of metals on potential efficiency of photosystem 2, PS2 (F_v/F_m) was negligible. The metal dependent reduction of PS2 quantum efficiency (_PS2) after plant adaptation in actinic irradiation was more noticeable. This could imply that reduced demand for ATP and NADPH in a dark phase of photosynthesis caused a down-regulation of PS2 photochemistry. Furthermore, in leaves of metal-treated plants the decrease in water percentage as well as lower contents of Chl and Fe were observed. Thus photosynthesis is not the main limiting factor for cucumber growth under Cu, Cd, or Pb stress.This revised version was published online in March 2005 with corrections to the page numbers.     

Elevated CO2 effects on carbon and nitrogen cycling in grass/clover turves of a Psammaquent soil

Effects of elevated CO₂ (525 and 700 L L^–1), and a control (350 L L^–1 CO₂), on biochemical properties of a Mollic Psammaquent soil in a well-established pasture of C3 and C4 grasses and clover were investigated with continuously moist turves in growth chambers over four consecutive seasonal temperature regimes from spring to winter inclusive. After a further spring period, half of the turves under 350 and 700 L L^–1 were subjected to summer drying and were then re-wetted before a further autumn period; the remaining turves were kept continuously moist throughout these additional three consecutive seasons. The continuously moist turves were then pulse-labelled with ¹⁴C-CO₂ to follow C pathways in the plant/soil system during 35 days.Growth rates of herbage during the first four seasons averaged 4.6 g m^–2 day^–1 under 700 L L^–1 CO₂ and were about 10% higher than under the other two treatments. Below-ground net productivity at the end of these seasons averaged 465, 800 and 824 g m^–2 in the control, 525 and 700 L L^–1 treatments, respectively.in continuously moist soil, elevated CO₂ had no overall effects on total, extractable or microbial C and N, or invertase activity, but resulted in increased CO₂-C production from soil, and from added herbage during the initial stages of decomposition over 21 days; rates of root decomposition were unaffected. CO₂ produced h^–1 mg^–1 microbial C was about 10% higher in the 700 L L^–1 CO₂ treatment than in the other two treatments. Elevated CO₂ had no clearly defined effects on N availability, or on the net N mineralization of added herbage.In the labelling experiment, relatively more ¹⁴C in the plant/soil system occurred below ground under elevated CO₂, with enhanced turnover of ¹⁴C also being suggested.Drying increased levels of extractable C and organic-N, but decreased mineral-N concentrations; it had no effect on microbial C, but resulted in lowered microbial N in the control only. In soil that had been previously summer-dried, CO₂ production was again higher, but net N mineralization was lower, under elevated CO₂ than in the control after autumn pasture growth.Over the trial period of 422 days, elevated CO₂ generally appears to have had a greater effect on soil C turnover than on soil C pools in this pasture ecosystem.     

Developmental aspects of long-term callus culture ofVicia faba L.

Summary Vicia faba callus line (VFS 1), isolated from expiants of immature embryo, grew satisfactorily onMurashige andSkoog complete medium with 1.38 M 2,4-D, or with 0.92 M 2,4-D to which 1.0 M kinetin was added. It also grew well on the B 5 modified medium containing 2.3 M 2,4-D and 25.0 M kinetin. On the last of these media the cultures grew more uniformly and without necrosis. They also showed diminishing variation in polyploidy in favour of diploids and corresponding aneuploids (hypodiploids).After being cultured for nearly three years on MS containing 1.38 M 2,4-D, 8–33% of cultures of VFS 1 were able to regenerate roots when transferred to either MS half strength with 5.37 M NAA, or to a medium without 2,4-D, or else to media with the addition of kinetin only (in various concentrations).     

Cadmium-copper antagonism in the activation of periplasmic nitrous oxide reductase of copper-deficient cells fromPseudomonas stutzeri

Summary Copper-deficient cells ofPseudomonas stutzeri strain ZoBell synthesize catalytically inactive nitrous oxide (N₂O) reductase which is activated by added Cu(II) in the absence of de novo protein synthesis. The apparentK _m for the activation process is 0.13 M. Activation is temperature-dependent and is inhibited by Cd(II)(K _i 1.27 M) and less strongly by Zn(II), Ni(II), and Co(II). The same metal ions at 20 M have little or no effect on N₂O reduction of intact cells. Apo-N₂O reductase of transposon Tn5-inducednos ^– mutants with defective Cu-chromophore biosynthesis is not reactivated by Cu(II). N₂O reductase of Cu-sufficient and Cu-deficient wild type, and ofnos ^– mutants is localized in the periplasm, the latter providing the likely site of metal incorporation into the apoenzyme.