Cilia regeneration in Tetrahymena : A simple reproducible method for producing large numbers of regenerating cells

We have developed an improved medium in which Tetrahymena can be deciliated by gentle shearing. The cells remain viable and regenerate a new complement of cilia. Unlike previous methods for viable deciliation of Tetrahymena, this method is easily adaptable to large numbers of cells, to cells in different stages of the life cycle (growing, starved, conjugating), and to both commonly studied species, T. thermophila and T. pyriformis. Starved T. thermophila deciliated by this method regained motility by 1 h, regenerated oral apparatus by 4.0 h and restored tubulin in cilia at a linear rate of about 3 pg h⁻¹ cell⁻¹.     

Comparison of axonemal proteins from two kinds of Tetrahymena. I. Different characteristics of dyneins in heat stability

Tetrahymena thermophila could still swim after incubation of the cell body at 40°C for 30 min, whereas Tetrahymena pyriformis did not show any motility after the treatment. Turbidity measurements revealed that axonemes of T. pyriformis lost ATP-dependent sliding activity by the heat treatment, whereas those of T. thermophilia still had the activity under the same conditions. In connection with this difference in susceptibility to high temperature, the biochemical characteristics of dyneins were compared between the two species of Tetrahymena. Axonemal dyneins from the two species had significant vanadate-sensitive ATPase activity even after the heat treatment. Native gel electrophoresis and the following two-dimensional electrophoresis showed that the outer arm dynein of T. thermophilia is more stable in maintaining native configuration than that of T. pyriformis against the heat treatment, although both treated dyneins keep three (α, β and γ) subunits. Analysis by peptide mapping demonstrated that β- and γ-subunits of the outer arm dynein are considerably different in amino acid sequences between the two species. These results imply that dynein of T. thermophilia changed their amino acid sequences and biochemical characteristics to adapt to high temperature.     

DNA replication in macronuclei of Tetrahymena pyriformis GL

DNA replication in macronuclei of Tetrahymena pyriformis GL has been studied to discriminate between hypotheses developed for the interpretation of intraclonal differentiation in ciliated protozoa (the diploid subnuclear, and the ‘master’-‘slave’ hypotheses). Tetrahymena cells were grown in a heavy ¹⁵N-³H medium and then transferred to a light ¹⁴N-¹⁴C medium. DNA was isolated after various periods following this transfer and studied in equilibrium CsCl density gradient centrifugation. Time-related changes in the DNA buoyant density pattern were investigated. The data obtained are interpreted to mean that all DNA in macronuclei of asynchronously growing Tetrahymena at exponential phase replicates semiconservatively once in a cell cycle. These data are in good agreement with the findings of Andersen & Zeuthen obtained on synchronous Tetrahymena cultures in the presence of BUdR.These results are not consistent with the ‘master’-‘slave’ hypothesis. The diploid subnuclear hypothesis is not in accord with other experimental evidence. An alternative hypothesis has been proposed concerning the nature of the macronuclear units and the process of determination. The two main points of this hypothesis are: (a) macronuclear units are diploid genome fragments (‘nucleosomes’); (b) determination is a process of haploidization by ‘allelic splitting’ at a definite macronuclear fission. Consistency with experimental data is discussed and some predictions of the hypothesis are given.     

Maturation dependent decline of adenylate cyclase in rabbit red blood cell membranes

Adenylate cyclase (EC 4.6.1.1) was studied in membrane preparations of reticulocyte-rich blood obtained from phenylhydrazine-treated rabbits and compared to that of untreated animals.Basal and fluoride-stimulated activities were decreased 2- and 4-fold, respectively, during the process of maturation.Catalytic parameters such as time course, protein, ATP, Mg²⁺ concentration curves and K_m have been determined and were found to be similar in the reticulocyte and the erythrocyte.Adenylate cyclase was sensitive to GTP, 5′-guanylyl imidodiphosphate, prostaglandin E₁ and prostaglandin E₂. Activation by prostaglandin E₁ was higher than that produced by prostaglandin E₂. Only additive effect was found when 5′-guanylyl imidodiphosphate or GTP was added to hormone-stimulated activity. The sensitivity of the enzyme to these effectors was decreased over the transition reticulocyte-erythrocyte.In either cell the enzyme was not activated by catecholamines (epinephrine, norepinephrine, isoproterenol).     

Purification and substrate characterization of α-ketobutyrate decarboxylase from Pseudomonas putida

α-Ketobutyrate decarboxylase encoded in the -methionine catabolism operon of Pseudomonas putida is homologous with the E1 component of pyruvate dehydrogenase complex from gram-negative bacteria. The enzyme was purified to homogeneity from the cell extract of an Escherichia coli transformant. The purified enzyme was homodimeric with a subunit of M_r 93,000 on SDS-PAGE. The enzyme activity was activated by the addition of both thiamine pyrophosphate (TPP) and a divalent cation, such as Mg²⁺, Mn²⁺, and Co²⁺. The enzyme showed high activity for α-ketobutyrate and α-keto-n-valerate rather than pyruvate, but the α-keto acids with increasing length of the side chain as well as branching, such as α-keto-n-caproate and α-keto-3-methylvalerate, were not used by the enzyme. The K_m values for α-ketobutyrate and pyruvate were 0.016 and 0.147 mM, respectively, and the k_cat/K_m value (10.69 s⁻¹ mM⁻¹) for α-ketobutyrate was 29-fold greater than that for pyruvate. Thus, α-ketobutyrate decarboxylase is distinguished from the pyruvate dehydrogenase E1 component with respect to the substrate specificity, although their structural and enzymological properties were similar. These results suggest that the unique substrate specificity of α-ketobutyrate decarboxylase is due to a slight difference in the highly conserved active sites of both enzymes.     

Cyclic 3′,5′-AMP phosphodiesterase in Physarum polycephalum: II. Kinetic properties

The effect of several inhibitors of the enzyme cyclic 3′,5′-AMP phosphodiesterase as chemoattractants in Physarum polycephalum was examined. Of the compounds tested, 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Roche 20-1724/001) and 1-ethyl-4-(isopropylidinehydrazino)-1H-pyrazolo-(3,4-b)-pyridine-5-carboxylic acid ethyl ester, hydrochloride (Squibb 20009) were the most potent attractants. 3-Isobutyl-1-methyl xanthine, theophylline, and morin (a flavanoid) were moderate attractants and sometimes gave negative chemotaxis at high concentrations. Cyclic 3′,5′-AMP was an effective, but not potent attractant. A repellent effect following the positive chemotactic action was sometimes observed with cyclic 3′,5′-AMP at concentrations as high as 1 · 10⁻² M. Dibutyryl cyclic AMP appeared to be a somewhat more potent attractant than cyclic 3′,5′-AMP. The 8-thiomethyl and 8-bromoderivatives of cyclic AMP, which are poorly hydrolyzed by the phosphodiesterase, were not attractants in Physarum. Possible participation of cyclic 3′,5′-AMP in the directional movement in P. polycephalum is discussed.     

Biochemical characterization of Rhodococcus erythropolis N′4 nitrile hydratase acting on 4-chloro-3-hydroxybutyronitrile

The Rhodococcus erythropolis strain (N′4) possesses the ability to convert 4-chloro-3-hydroxybutyronitrile into the corresponding acid. This conversion was determined to be performed by its nitrile hydratase and amidase. Ammonium sulfate fractionation, DEAE ion exchange chromatography, and phenyl chromatography were used to partially purify nitrile hydratase from cell-free extract. A SDS-PAGE showed that the partially purified enzyme had two subunits and gel filtration chromatography showed that it consisted of four subunits of α₂β₂. The purified enzyme had a high specific activity of 860 U mg⁻¹ toward methacrylonitrile. The enzyme was found to have high activity at low temperature range, with a maximum activity occurring at 25 °C and be stable in the presence of organic acids at higher temperatures. The enzyme exhibited a preference for aliphatic saturated nitrile substrates over aliphatic unsaturated or aromatic ones. It was inhibited by sulfhydryl, oxidizing, and serine protease inhibitors, thus indicating that essential cysteine and serine residues can be found in the active site.The purified nitrile hydratase was able to convert 4-chloro-3-hydroxybutyronitrile into the corresponding amide at 15 °C. GC analysis showed that the initial conversion rate of the reaction was 215 mg substrate consumed min⁻¹ mg⁻¹. This demonstrated that this enzyme could be used in conjunction with a stereoselective amidase to synthesize ethyl (S)-4-chloro-3-hydroxybutyrate, an intermediate for a hypercholesterolemia drug, Atorvastatin.     

Gastric HCO3-stimulated ATPase: Evidence against its microsomal localization in rat fundus mucosa

Rat gastric mucosa was shown to contain a Mg²⁺-dependent ATPase which is stimulated by HCO₃⁻ at pH 8–9.Triton X-100 solubilizes this HCO₃⁻-stimulated, Mg²⁺-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3).The gastric mucosa was resolved into five subcellular fractions by differential centrifugation. A large granule fraction (Fraction M), 28 000 g · min, was characterized by cytochrome c oxidase (marker enzyme for mitochondria). A microsomal fraction (Fraction P), 2 760 000 g · min, was characterized by 5′-nucleotidase(5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) (plasma membrane).The Mg²⁺-dependent ATPase was demonstrated to have a bimodal mitochondrial membranous localization: 24% of its activity is associated with cytochrome c oxidase, and 75% with 5′-nucleotidase(5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) at pH 8.The HCO₃⁻ addition resulted in two opposite effects: (1) a strong stimulation (84%) in Fraction M; (2) a slight inhibition (12%) in Fraction P.Fraction M was subfractionated by equilibration on a sucrose gradient. It gave rise to a homogeneous mitochondrial (d, 1.17–1.21) Mg²⁺-dependent ATPase, closely associated with cytochrome c oxidase. This ATPase is strongly stimulated (×2) by HCO₃⁻. The subfractionation of Fraction P gave rise to two distinct ATPases: (1) the major one is associated with membranous (d, 1.10–1.15) material marked by 5′-nucleotidase and is slightly inhibited by HCO₃⁻; (2) the other is associated with denser (d, 1.17–1.21) material and is stimulated by HCO₃⁻.The bicarbonate-stimulated fraction of the Mg²⁺-dependent ATPase activity found in the gastric microsomal fraction is assumed to arise from mitochondrial cross-contamination. Further support comes from the optimal HCO₃⁻ concentration. In addition, SCN⁻ is shown to specifically inhibit the ATPase of Fraction M.From these results it appears that the implication of HCO₃⁻-stimulated ATPase in the gastric secretion of H⁺ is not as clear as had been suggested. However, in the view of an ATPase-supported model for H⁺ secretion, attention can be directed towards the Mg²⁺-dependent ATPase found to be associated with microsomes.     

Proteolytic degradation of ribosomal proteins during isolation of ribosomes and ribosomal subunits from Tetrahymena

A preparation procedure previously used to isolate active ribosomal subunits from an amicronucleate strain of Tetrahymena of undefined phenoset (T. «pyriformis CGL) yields inactive subunits when applied to other amicronucleate or to micronucleate strains of this protozoa.Proteolytic degradation of a small number of ribosomal proteins during preparation of ribosomal subunits from these strains explains this results. If cell extraction and ribosome isolation are carried out in the presence of iodoacetamide, proteolytic activity is inhibited and active ribosomal subunits are obtained. Comparison of the protein complements of active ribosomal subunits prepared in the presence of iodoacetamide from three amicronucleate strains of Tetrahymena reveals small but significant differences.     

Phospholipase D Activity in the Tetrahymena pyriformis GL

Phospholipase D (PLD) is an enzyme which participates in the signalling mechanism cleaving phosphatidylcholine (PC) to choline and phosphatidic acid (PA). In Tetrahymena pyriformis GL this enzyme activity is enhanced by different kinds of agonists (sodium orthovanadate, sodium fluoride and phorbol 12-myristate 13-acetate), and its activity can be inhibited by inhibitors such as pertussis toxin, calphostin C, genistein, trifluoperazine. These results suggest that the PLD signalling pathway is connected with the tyrosine kinase, phospholipase C, phosphatidylinositol and G-protein coupled signalling pathways. By demonstrating the PLD activity in Tetrahymena our knowledge on the signalling mechanisms at a unicellular level has been extended. The results support our view that most transducing mechanisms that are characteristic of mammalian cells are also in the protozoan Tetrahymena. © 1997 John Wiley & Sons, Ltd.     

The α1-adrenergic receptor in human erythrocyte membranes mediates interaction in vitro of epinephrine and thyroid hormone at the membrane Ca-ATPase

Membrane Ca²⁺-ATPase activity was stimulated in vitro separately by T₄ (10⁻¹⁰ M) and by epinephrine (10⁻⁶ M). In the presence of a fixed concentration of T₄, additions of 10⁻⁸ and 10⁻⁶ M epinephrine reduced the T₄ effect on the enzyme. β-Adrenergic blockade with propranolol (10⁻⁶ M) prevented stimulation by epinephrine of Ca²⁺-ATPase activity, but did not prevent the suppressive action of epinephrine on T₄-stimulable Ca²⁺-ATPase. In contrast α₁-adrenergic blockade with unlabelled prazosin restored the effect of T₄ on Ca²⁺-ATPase activity in the presence of epinephrine. Like propranolol, prazosin prevented enhancement of enzyme activity by epinephrine in the absence of thyroid hormone. Neither prazosin nor propranolol had any effect on the stimulations by T₄ of red cell Ca²⁺-ATPase in the absence of epinephrine. Analysis of radiolabelled prazosin binding to human red cell membranes revealed the presence of a single class of high-affinity binding sites (K_d, 1.2 × 10⁻⁸ M; B_max, 847 fmol/mg membrane protein). Thus, the human erythrocyte membrane contains α₁-radrenergic receptor sites that are capable of regulating Ca²⁺-ATPase activity.     

Effects of NaCl and GTP on the inhibition of platelet adenylate cyclase by 1-O-octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (synthetic platelet-activating factor)

We have investigated the effects of NaCl and GTP on the inhibition of platelet adenylate cyclase by 1-O-octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (1-octadecyl-2-acetyl-G-3-PC), using particulate fractions from human and rabbit platelets that had been frozen and thawed in the presence of ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetate to prevent Ca²⁺-dependent proteolysis. When 10 μM GTP was present, 100 mM NaCl stimulated the activity of the rabbit enzyme 5.6-fold and that of the human enzyme 2.2-fold. Under these conditions, maximum inhibitions of 90% and 64% were obtained on addition of 100 nM 1-octadecyl-2-acetyl-G-3-PC to rabbit and human preparations, respectively. These inhibitions resulted partly from an NaCl-independent inhibition of basal enzyme activity and partly from reversal of the stimulatory effect of NaCl. The relative abilities of the chlorides of different monovalent cations to enhance inhibition of rabbit platelet adenylate cyclase were: NaCl >LiCl >KCl >choline chloride. NaCl also increased the concentrations of 1-octadecyl-2-acetyl-G-3-PC required for half-maximal inhibition of adenylate cyclase but this action of NaCl did not correlate with its stimulatory effect on enzyme activity. After particulate fractions from platelets of either species were washed, 10 μM GTP inhibited basal adenylate cyclase activity in the absence of NaCl but stimulated the enzyme in the presence of NaCl. Inhibition of adenylate cyclase by 1-octadecyl-2-acetyl-G-3-PC was then either enhanced by GTP (rabbit material) or completely dependent on added GTP (human material). Stimulation of the activity of the washed human preparations by NaCl required GTP, but concentrations lower than required for potentiation of the inhibitory effect of 1-octadecyl-2-acetyl-G-3-PC by NaCl were effective.     

P2X<Subscript>7</Subscript> Receptor Stimulation of Membrane Internalization in a Thyrocyte Cell Line

Using fluorescent membrane markers, we have previously shown that extracellular ATP stimulates both exocytosis and membrane internalization in the Fisher rat thyroid cell line FRTL. In this study, we examine the actions of ATP using whole-cell recording conditions that favor stimulation of membrane internalization. ATP stimulation of the P2X₇ receptor activated a reversible, Ca²⁺-permeable, cation conductance that slowly increased in size without changes in ion selectivity. ATP also induced a delayed irreversible decrease in cell capacitance (C_m) that was equivalent to an 8% decrease in membrane surface area. Addition of guanosine 5′-0-2-thiodiphosphate to the pipette solution inhibited the ATP-induced decrease in C_m without affecting channel activation. The effects of ATP on membrane conductance were mimicked by 2′,3′-O-(4-benzoylbenzoyl)-ATP, but not by UTP, adenosine, or 2-methylthio-ATP, and were inhibited by pyridoxal phosphate-6-azophenyl-2′4′-disulfonic acid, adenosine 5′-triphosphate-2′3′-dialdehyde, and Cu²⁺. The capacitance decrease persisted in Na⁺-, Ca²⁺- and Cl⁻-free external saline or with Ca²⁺-free pipette solution. It is concluded that ATP activation of the inotropic P2X₇ receptor stimulates membrane internalization by a mechanism that involves intracellular GTP, but does not require internal Ca²⁺ or influx of Na⁺ or Ca²⁺ through the receptor-gated channel.     

Bacterial Expression and Purification of the Arabidopsis NADPH–Cytochrome P450 Reductase ATR2

An N-terminally modified form of the Arabidopsis NADPH–cytochrome P450 ATR2 (ATR2mod) was expressed from the tactac promoter in Escherichia coli to obtain high yields of the enzyme. The N-terminal modification eliminates the predicted chloroplast transit peptide of ATR2 allowing for more efficient expression. ATR2mod was purified from membrane extracts using a 2′,5′-ADP–agarose affinity column. The specific activity of the purified ATR2mod for cytochrome c reduction was 9.4 μmol min⁻¹ mg⁻¹ and the K_m for cytochrome c reduction was 15 ± 2 μM. The purified NADPH–cytochrome P450 reductase was able to support function of CYP79B2.     

ATP increases chemoattractant induced cyclic GMP accumulation in Dictyostelium discoideum

Changes in guanosine cyclic 3′,5′-monophosphate associated with adenosine cyclic 3′,5′-monophosphate and folic acid addition in the presence of ATP have been examined in Dictyostelium discoideum. Preincubation with 1 mM ATP had no effect on the basal cyclic GMP level but increased the cycli GMP accumulation in response to cylci AMP (5·10⁻⁸ M) or folic acid (5·10⁻⁶ M) 40–50%. ATP could not be replaced by ADP of 5′-adenylyliminodiphosphate. Because ATP has no effect on cyclic AMP receptor binding these results indicate that structural membrane alterations (e.g. membrane phosphorylation) may control the transduction of a chemotactic signal.     

Membrane reconstitution in CHL-R mutants of Escherichia coli K 12 V. ATPase incorporation into particles formed by complementation

Mechanical treatments of cell suspensions of Escherichia coli K 12 strain PA 601, and its two mutants chl A⁻ and chl B⁻, in a buffer without Mg²⁺ lead to partial solubilization of membrane-bound ATPase. After ultracentrifugation of cell-free extracts, ATPase can be recovered in the soluble fraction. Contrary to membrane ATPase, the soluble enzyme has the following properties: (1) it is insensitive to N,N′-dicyclohexylcarbodiimide; (2) heat-inactivation kinetics show a reactivation in the first 3 min and the half-time is 15 min; (3) ADP is a substrate. In the course of complementation between soluble fractions of mutants chl A⁻ and chl B⁻, a part of soluble ATPase is incorporated into the newly formed particles. The specific activity of these particles is nearly the same as that of native particles; the ATPase bound to native membrane and the ATPase bound to the newly-formed particles both have the same biochemical properties.     

High-performance liquid chromatographic method for determination of DDT and its degradation products in rat plasma, liver and brain: validation and application to a pharmacokinetic study

A sensitive and reliable high-performance liquid chromatographic (HPLC) method, using a solid-phase extraction (SPE), was established and validated for determination of p,p′-DDT [1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane] and its metabolite p,p′-DDE [1,1-(2,2-dichloroethanylidene)-bis(4-chlorobenzene)] in rat plasma, liver and brain. After being diluted with water, plasma, liver and brain samples were applied to a solid-phase extraction C₁₈ cartridge. The extraction containing p,p′-DDT and p,p′-DDE from the cartridge were cleaned-up using a Florisil Sep-Pak cartridge. The samples were analyzed by HPLC using UV detection at 238 nm. The limit of detection for p,p′-DDT and p,p′-DDE was 0.1 mg kg⁻¹ liver or brain and 0.1 mg l⁻¹ plasma. For six replicate samples at 40, 4 and 0.2 mg kg⁻¹, intra-day precision values were within 4.9% for plasma, 6.4% for liver, and 9.7% for brain. Inter-day precision values at 4 mg kg⁻¹ were within 8.2% for plasma and tissues. The method performances were shown to be selective for p,p′-DDT and p,p′-DDE, and linear over the range 0.04–12 mg kg⁻¹ (mg l⁻¹ for plasma). The absolute recoveries of p,p′-DDT and p,p′-DDE in rat plasma and tissues were over 92%. The method was proved to be applicable to the pharmacokinetic study of DDT in rats after a single oral administration.     

Steric and electronic properties of the cofactor's amino group control the lifetime of the central carbanion/enamine intermediate in transketolase

Transketolase (TK), a thiamin diphosphate (ThDP) dependent enzyme, catalyzes the reversible transfer of a two-carbon unit from keto- to aldo-substrates. Dihydroxyethylthiamin diphosphate (DHEThDP), formed as a result of cleavage of the donor substrate, serves as an intermediate of the TK reaction. TK from the yeast Saccharomyces cerevisiae is unique among thiamin enzymes displaying enzymatic activity after reconstitution with a methylated analogue of the native cofactor, 4′-methylamino-ThDP. The reconstitution of the apoenzyme with both ThDP and the methylated analogue can be analyzed by near UV circular dichroism. It was demonstrated that in the native holoenzyme and in the complex of TK with 4′-methylamino-ThDP the formation of the dihydroxyethyl-based carbanion/enamine took place with comparable rate constants, whereas the protonation of the reactive species was much faster in the complex with the analogue. The enzymatic activity of the enzyme reconstituted with 4′-methylamino-ThDP was 10fold higher in the ferricyanide assay. We suggest that a methylation of the 4′-amino group of ThDP impairs the resonance stabilization of the carbanion/enamine intermediate both sterically and electronically, thus allowing either a faster protonation or oxidation reaction by ferricyanide. The formation of the optically active DHE-4′-methylamino-ThDP was monitored by near UV circular dichroism spectra and corroborated by ¹H NMR analysis. The protonated form of the intermediate DHE-4′-methylamino-ThDP was released from the active sites of TK and accumulated in the medium on preparative scale.     

Effects of 4-acetamido-4′-isothiocyano-2,2′-disulfonic stilbene on ion transport in turtle bladders

The disulfonic stilbene (4-acetamido-4′-isothiocyano-2,2′-disulfonic stilbene) is found to be more potent than acetazolamide as an anion transport inhibitor in the turtle bladder, but less potent than acetazolamide as a carbonic anhydrase inhibitor. The anion-dependent (HCO₃^-−, Cl⁻) moeity of the short-circuiting current is eliminated by 4-acetamido-4′-isothiocyano-2,2′-disulfonic stibene, but only after its addition to the serosal bathing fluid. Whereas 4-acetmido-4′-isothiocyano-2,2′-disulfonic stilbene has no effect om Na⁺transport across the bladder, it is more potent than ouabain as an inhibitor of microsomal (Na⁺+K⁺)-ATPase of both turtle bladder and eel electric organ.     

PAF-antagonistic bicyclo[3.2.1]octanoid neolignans from leaves of Ocotea macrophylla Kunth. (Lauraceae)

Di-nor-benzofuran neolignan aldehydes, Δ⁷-3,4-methylenedioxy-3′-methoxy-8′,9′-dinor-4′,7-epoxy-8,3′-neolignan-7′-aldehyde (ocophyllal A) 1, Δ⁷-3,4,5,3′-tetramethoxy-8′,9′-dinor-4′,7-epoxy-8,3′-neolignan-7′-aldehyde (ocophyllal B) 2, and macrophyllin-type bicyclo[3.2.1]octanoid neolignans (7R, 8R, 3′S, 4′S, 5′R)-Δ⁸′-4′-hydroxy-5′-methoxy-3,4-methylenedioxy-2′,3′,4′,5′-tetrahydro-2′-oxo-7.3′,8.5′-neolignan (ocophyllol A) 3, (7R, 8R, 3′S, 4′S, 5′R)-Δ⁸′-4′-hydroxy-3,4,5′-trimethoxy-2′,3′,4′,5′-tetrahydro-2′-oxo-7.3′,8.5′-neolignan (ocophyllol B) 4, (7R, 8R, 3′S, 4′S, 5′R)-Δ⁸′-4′-hydroxy-3,4,5,5′-tetramethoxy-2′,3′,4′,5′-tetrahydro-2′-oxo-7.3′,8.5′-neolignan (ocophyllol C) 5, as well as 2′-epi-guianin 6 and (+)-licarin B 7, were isolated and characterized from leaves of Ocotea macrophylla (Lauraceae). The structures and configuration of these compounds were determined by extensive spectroscopic analyses. Inhibition of platelet activating factor (PAF)-induced aggregation of rabbit platelets were tested with neolignans 1–7. Although compound 6 was the most potent PAF-antagonist, compounds 3–5 showed some activity.