Similarly to Escherichia coli K-12 IAM 1264, trans-4-guanidinomethylcyclohexanecarboxylic acid 4-tert-butylphenyl ester (GMCHA-OPhtBu), a trypsin inhibitor, had a strong inhibitory effect on the growth of various strains of E. coli K-12, such as W 3350, AB 1157, JM 103, W 3110, C 600r-m- and C 600r+m+, preceded by suppressive effects of GMCHA-OPhtBu on DNA synthesis in these strains, although the inhibitory effects varied from strain to strain. These results suggested the possible involvement of a trypsin-like proteinase in DNA synthesis. A trypsin-like proteinase was partially purified from E. coli K-12 AB 1157 by ammonium fractionation and successive chromatographies on DEAE-cellulose, Sephadex G-100 and arginine-Sepharose 4B columns. The properties were compared with those of proteinase In, which instantly appears just before the onset of DNA synthesis and seems to participate in the initiation of DNA replication, and which was purified from E. coli K-12 IAM 1264. The proteinase from E. coli K-12 AB 1157 was identified with proteinase In. These results suggest that proteinase In must be widely distributed in various E. coli strains and plays a pivotal role in the onset of DNA replication.
A plasmid for direct expression in Escherichia coli of the mature form bovine adrenodoxin reductase was constructed from the full-size cDNA for the enzyme [Y. Sagara, Y. Takata, T. Miyata, T. Hara, and T. Horiuchi, J. Biochem. (Tokyo), 102, 1333 (1987)] and an expression vector pCWori+. The recombinant adrenodoxin reductase was purified from the transformed E. coli cell lysates using adrenodoxin-Sepharose affinity chromatography [T. Sugiyama and T. Yamano, FEBS Lett., 52, 145 (1975)] with a yield of 2.5mg/l of culture. The purified recombinant enzyme showed a single band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and migration was identical with that of the authentic enzyme purified from bovine adrenal cortex mitochondria. The recombinant enzyme had Ser at its amino-terminus and the sequence of the amino terminal 9 residues was identical with that of the authentic bovine enzyme. The absorption spectrum of the recombinant enzyme showed peaks at 270, 376, and 450 nm and shoulders at 425 and 475 nm. Flavin content of the recombinant enzyme was 0.8 mol FAD/mol. The apparent Km value for bovine adrenodoxin in NADPH-cytochrome c reductase activity using a reconstitution system was 16 nM, a value comparable with that of the authentic bovine enzyme (17 nM). The cholesterol side chain cleavage activity with a reconstitution system was about 75% of that obtained when the authentic enzume was used.
The structural alteration of human fibrinogen (Fbg) by cis-diamminedichloroplatinum(II)(cis-DDP) was investigated using intrinsic fluorescence and circular dichroism (CD) spectra. In a preceding report, incubation of Fbg with cis-DDP under physiological conditions resulted in the cleavage of disulfide (S-S) bonds (N. Ohta, T. Yotsuyanagi and K. Ikeda, J. Pharmacobio-Dyn., 15, 611 (1992)). The intensity of fluorescence for cis-DDP-treated Fbg, in which 6.7 S-S bonds per mol of protein were cleaved, was reduced to 42% relative to native Fbg. CD studies suggested that the α-helix content decreased from 42% in its natural state to 29% in the cis-DDP-treated form. The extent of the fluorescence quenching and the decrease in helix correlated with the number of cleaved S-S bonds. The results indicate that cis-DDP leads to a secondary conformational alteration of Fbg involving a structural perturbation of nearby tryptophan residue(s) through S-S bond cleavage. In a buffer containing calcium, however, the smaller fluorescence quenching and the smaller helix decline were observed in cis-DDP-treated-Fbg, even though the same level of S-S cleavage occurred. Calcium binding would protect Fbg in terms of structural alteration as well as S-S cleavage by both dithiothreitol (DTT) and cis-DDP.
Following oral dosing of [14C]clentiazem to rats the metabolites in urine and bile were separated and their chemical structures were investigated by HPLC and GC-MS analyses. Fifteen basic, 6 acidic, 2 neutral and 4 conjugated metabolites were found in urine and/or bile.Eight basic metabolites (MB1-8) were identified as the synthetic compounds; deacetyl clentiazem (MB1), N-monodemethyl clentiazem (MB2), deacetyl-N-monodemethyl clentiazem (MB3), deacetyl-O-demethyl clentiazem (MB4), N-monodemethyl-O-demethyl clentiazem (MB5), deacetyl-N-monodemethyl-O-demethyl clentiazem (MB6), O-demethyl clentiazem (MB7) and N-didemethyl clentiazem (MB8). The chemical structures of seven basic metabolites (MB9-15) were assigned as follows, deacetyl-N-didemethyl'clentiazem (MB9), O-demethyl-N-didemethyl clentiazem (MB10), deacetyl-O-demethyl-N-didemethyl clentiazem (MB11), N-monodemethyl-2-hydroxy-methoxyphenyl clentiazem (MB12), deacetyl-2-hydroxy-methoxyphenyl clentiazem (MB13), deacetyl-N-monodemethyl-2-hydroxy-methoxyphenyl clentiazem (MB14) and deacetyl-N-didemethyl-2-hydroxy-methoxyphenyl clentiazem (MB15).Four acidic metabolites were identified as the synthetic compounds : (+)-(2S, 3S)-3-(acetyloxy)-8-chloro-3, 4-dihydro-2-(4-methoxyphenyl)-4-oxo-1, 5-benzothiazepin-5(2H)-acetic acid (MA1), deacetyl-MA1 (MA2), O-demethyl-MA1 (MA3) and deacetyl-O-demethyl-MA1 (MA4); and the two remaining acidic metabolites, MA5 and MA6, were presumed to be hydroxylated MA3 and MA4, respectively.Two neutral metabolites were identified as the synthetic compounds; (+)-(2S, 3S)-3-(acetyloxy)-8-chloro-3, 4-dihydro-2-(4-methoxyphenyl)-4-oxo-1, 5-benzothiazepin-5(2H)-acetonitrile (MN1) and deacetyl MN1 (MN2). Other two metabolites conjugated with glucuronic acid were found in bile and the structures were presumed to be 8-chloro-2, 3-dihydro-3-hydroxy-5-(2-hydroxyethyl)-2-(4-hydroxyphenyl)-1, 5-benzothiazepin-4(5H)-one (MN3) and 2-methoxyphenyl MN3 (MN4). The glucuronide or sulfate of MA4 was also detected.These metabolites were formed by a number of pathways including deacetylation, deamination, N-demethylation, O-demethylation, aromatic hydroxylation and conjugation.
The plasma concentrations and time courses of radioactivity and unchanged drug, the excretion of radioactivity into urine and feces, and the proportion of metabolites in plasma and urine were studied after oral administration of [14C]clentiazem to male and female rats and dogs.Apparent sex-related differences were found in the disposition and metabolism of clentiazem in rats. The plasma levels of radioactivity and acidic metabolites were higher in males than in females. The plasma levels of unchanged drug, on the other hand, were about the same in both sexes.Higher conversion of clentiazem to its acidec metabolites in the liver of male rats and higher excretion of the acidic metabolites in the urine of female rats, presumably due to sex-related differences in cytochrome P-450 and renal clearance, respectively, seem to explain these differences in the disposition of clentiazem in male and female rats.No suggestion of a similar sex difference was found in dogs. The plasma concentrations and time courses of radioactivity and unchanged drug in male dogs were similar to those in female dogs, and the excretion of radioactivity in both sexes was also similar. The main plasma metabolite in male and female dogs was O-demethyl clentiazem (MB7).A species difference between rat and dog was suggested, since the major metabolic pathways were different and no sex difference was found in dogs.
The effect of a new zinc compound, β-ananyl-L-histidinato zinc (AHZ), on osteopenia was investigated in rats with adjuvant arthritis. Arthritis was induced in female rats by administering 1% Mycobaterium butyricum (MB) into the subplantar surface of the right hind paw. AHZ (10, 30 and 100 mg/kg body weight) was orally administered to MB-treated rats 28 times at 24-h intervals, and the rats were bled 24 h after the last administration. Treatment with MB caused a ramrkable increase in paw volume and a corresponding decrease in the ratio of albumin per globulin in serum, indicating taht the treatment induces inflammation. These alteration swere not significantly changed by the administration of AHZ (10, 30 and 100 mg/kg). Serum calcium and zinc concentrations are significantly decreased in rats with adjuvant arthritis. These decreases were completely restored by the administration of AHZ (30 and 100 mg/kg). Furthermore, the inflammation-induced decreases in alkaline phosphatase activity and calcium content in the femoral diaphysis were clearly blocked by the administration of AHZ (30 and 100 mg/kg). Also, the larger doses of AHZ (30 and 100 mg/kg) produced a significant increase in femoral-diaphyseal deoxyribonucleic acid and in the zinc content in rats with adjuvant arthritis. These results suggest that AHZ has a stimulating effect on bone formation in the femoral diaphysis of rats with adjuvant arthritis, although the compound did not have an anti-arthritic effect.
Changes were examined in myocardial catecholamine content and α1- and β1-adrenoceptors during the development of cardiomyopathy in Syrian hamsters (Bio 14.6) and age-matched healty controls. In addition, the effects of bunazosin, atenolol, xamoterol, ketanserin, and verapamil on the catecholamine content and [3H]prazosin, [3H]CGP12177 bindings to α1- and β1-adrenergic receptors of myocardium were compared with those of the controls. (1) Lower norepinephrine and dopamine levels were observed in 35-week-old cardiomyopathic hamster hearts than in the controls. There was, however, a tendency for a slight decrease of α1- and β1-adrenoceptors in cardiomyopathic hamsters. (2) Administration of bunazosin induced lower dopamine values in 18-week-old cardiomyopathic hamsters. (3) Xamoterol induced a higher Kd value for β1-adrenoceptors and lower dopamine content than for those with cardiomyopathy. Ketanserin also induced a higher Bmax value than for cardiomyopathy. Thus, drug treatments clearly change catecholamine content and binding characteristics of the adrenoceptors which play an important role in the development of cardiac hypertrophy and heart failure.
When erythrocyte membranes were incubated with adriamycin (ADM) in the presence of ferritin, lipid peroxidation occurred wiht release of iron from the ferritin. In the presence of apoferritin, ADM did not cause lipid peroxidation. Deferoxamine inhibited the ADM-induced lipid peroxidation in the presence of ferritin. These results indicate that lipid peroxidation depends upon the release of iron from ferritin. Even when the iron content in ferritin was very low, ADM could induce lipid peroxidaton. Superoxide dismutase, catalase and hydroxyl radical scavengers did not substantially affect lipid peroxidation, indicating that the peroxidation reaction was independent of superoxide, H2O2 and hydroxyl radicals. Ceruloplasmin, a ferroxidase, markedly inhibited lipid peroxidation but did not affect the release of iron from ferritin. ADM-Fe3+-binding erythrocyte membranes were readily formed during the incubation of erythrocyte membranes with ADM in the presence of ferritin, and deferoxamine removed iron from the ADM-Fe3+-binding membranes, indicating that the iron moiety of the ADM-Fe3+ complex is exposed at the membrane surface. These results may suggest that the peroxidation reaction occurs in a site-specific manner.
The effects of the antimalarial agent, (±)-erythro-mefloquine and related compounds [(±)-threo-mefloquine, (±)-erythro-N-methylmefloquine and its N-oxide, quinine, WR 184806 and halofanthrine] on the isolated mouse phrenic nerve diaphragm preparation were investigated. Based on their pharmacological effects, these compounds may be divided into two groups. The group I compounds, comprising (±)-erythro-mefloquine, (±)-threo-mefloquine and WR 184806, were found to exert a contractile effect on the muscle and also to inhibit the indirectly (nerve) stimulated and directly (muscle) stimulated (after α-bungarotoxin) twitch responses. The group II compounds, comprising the other compounds except halofanthrine, lacked a contractile effect on muscle but potentiated the directly stimulated twitch responses (after α-bungarotoxin). Halofanthrine did not elicit any response from the preparation.The minimum energy conformations of these compounds were determined using an interactive molecular modeling program which incorporates MMX force field for molecular mechanics calculations. Conformational analyses of the erythro and threo isomers of mefloquine hydrochloride were also undertaken using 1H-NMR. The 1H-NMR data supported the proposal made on the basis of MMX calculations that the erythro isomer exists in solution as one predominant conformer whereas the threo isomer is present in solution as a mixed population of two stable conformers. The structure-activity relationship of the compounds is discussed.
Formulations consisting of egg albumin, indomethacin (IND), and olive oil or fatty acids, were prepared by vigorous stirring using a high-speed homogenizer and subsequent freeze-drying. To confirm the anti-inflammatory properties and ulcerogenic effects of the formulatios, we examined the action of the formulations on carrageenan-induced edema in rats as well as their ulcerogenic actions in the same species. Compared with IND alone, albumin-IND-olive oil (9 : 1 : 4.3), albumin-IND-linolenic acid (9 : 1 : 4.3), albumin-IND-liolic acid (9 : 1 : 4.3), albumin-IND-oleic acid (9 : 1 : 4.3), albumin-IND-stearic acid (9 : 1 : 4.3), and albumin-IND-tristearin (9 : 1 : 4.3) formulations all exhibited a more potent inhibitory effect on carrageenan-induced edema. In addition, the inhibitory effects on edema formation of an albumin-IND (9 : 1) complex was as strong as that of IND alone. These results suggested that the biovailability of IND was increased by olive oil, fatty acid, and tristearin as absorbefacient agent. The increase in the bioavailability was evident from the fact that the mean plasma levels, maximum plasma levels (Cmax), and area under plasma concentration-time curve (AUC) values after oral administration of the albumin-IND-olive oil (9 : 1 : 4.3) formulation was significantly greater than that after administration of the drug alone. With respect to their ulcerogenic properties, the formulations were significantly less active than IND alone, suggesting that a reduction in the ulcerogenic activity of IND was by produced complexation with egg albumin.
To confirm the increased bioavailability of indomethacin (IND) when incorporated in a preparation with egg albumin and olive oil, we studied the detailed pharmaceutical characteristics of a ternary formulation consisting of egg albumin, IND and olive oil. From the results of X-ray powder diffraction measurements, the drug in the formulation was found to be in an amorphous form. When orally administered to rats, the ternary formulation significantly increased the plasma concentration and cumulative biliary and urinary excretion of IND alone as well as the urinary excretion of its major metabolite, desmethylindomethacin, compared with the drug alone. In addition, the dissolution rate of IND from the formulation was higher than that of the drug alone. These results clearly suggest that the bioavailability of IND was markedly improved by incorporating it in a protein-drug formulation containing olive oil as an absorbefacient element, and this effect may be due to an increased absorption of IND.
This study was carried out to compare the accelerative effect in ether extracts of "Koushikon" and "Nanshikon" on proliferation of granuloma tissue in rats, and to elucidate this effect on optical isomer of naphthoquinone derivatives in those extracts. The content of total naphthoquinone derivatives in the ether extracts of Koushikon and Nanshikon were found to be 56.1% and 25.4%. Among naphthoquinone derivatives, Koushikon contained mostly acetyl derivative and Nanshikon mostly teracryl derivative. The percentage of R-type (shikonin-type) in total naphthoquinone derivatives of the extracts was 85.5% and 3.8%. Each ether extract showed a dose-dependent acceleration on the cotton pellet-induced granuloma formation. Comparison with corresponding doses containing the same quantity of naphthoquinone derivatives showed the accelerative potency of ether extracts of Koushikon and Nanshikon to be about the same. The result suggests that the accelerative effect on proliferation of granuloma tissue depends primarily on the total content of naphthoquinone derivatives, and not on the ratio of the optically active isomers.
The lymphocyte stimulation test (LST) is one of the most useful laboratory tests for the identification of allergy to a specific drug. The present study was conducted to examine utility of the LST using the [3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] (MTT) assay as a method for diagnosing drug allergy. The basic experimental conditions for the MTT assay were determined by using CTLL cells from human T-lymphoma, dependent on IL-2, and human peripheral blood lymphocytes. The amount of MTT needed was 0.5 mg per well and the peak maximum of MTT formazan was close to 565 nm. The blastogenesis of the lymphocytes was expressed as a Stimulation Index (SI). The value of the SI was almost constant at cell numbers ranging from 5×105 to 1×106 cells/ml. The SI of some drugs using normal peripheral blood lymphocytes was less than 1.3.We used the MTT assay in the LST to examine the allergenicity of 83 drugs which were used in 43 cases of suspected drug-induced hepatitis. The range of SI was 0.92-2.02. An SI of 1.2 or more was seen in 62.8% of cases, 1.3 or more in 51.2% of cases, and 1.4 or more in 41.9% of cases. There were 26 drugs with SI greater than 1.3. Seven of these were antibiotics, while the rest included antihypertensives, analgesics, psychotropics, antiallergics and antiepileptic.This method is extremely well suited for the diagnosis of drug allergy in a clinical situaiton. The greatest benefit of the MTT assay is that the complete test procedures can be carried out in a general laboratory.
The effects of 1-geranylazacycloheptan-2-one (GACH) on the in vitro skin penetration of seven drugs with various lipophilicities were studied. The penetration of drugs from aqueous vehicle through the guinea pig skin was increased depending on the pretreatment dose of GACH. The largest enhancement was observed for 5-fluorouracil (5-FU) and a bell-shaped relationship was obtained between enhancement ratio and the octanol/water partition coefficients of drugs. Further, Laplace transformed equaiotns for percutaneous absorption of drug were derived from Fick's second law based on a two-layer skin diffusion model with polar and nonpolar routes in the stratum corneum. Through curve-fitting of their equations to penetration profiles using a nonlinear regression program MULTI(FILT) combined with a fast inverse Laplace transform (FILT) algorithm, the action mechanism of GACH was discussed in terms of its effect on the partitioning and diffusivity of drugs in each domain. With an increase in pretreatment dose of GACH, the estimated partition parameters of drugs into the nonpolar route increased but their diffusivities were little affected. The analysis based on a linear free-energy relationship suggested that the increase in partitioning of drugs into the nonpolar route was explained by its increasing polarity with GACH pretreatment.
Nineteen isoflavones were synthesized and their attracting activity to Aphanomyces euteiches zoospore were investigated. Isoflavones (1, 2, 4, 16 and 19) with an unsubstituted B ring were synthesized by the oxidation of corresponding 2'-hydroxychalcones with thallium (III) nitrate trihydrate in methanolic perchloric acid. A hydroxyl group at the C-5 position in isoflavones was necessary for strong attraction to A. euteiches zoospore. The introduction of an additional hydroxyl group at the C-7 or C-4' position strengthened the attracting activity. Moreover, the methylation of the C-7 hydroxyl group strengthened the attracting activity, but the methylation of the C-4' hydroxyl group slightly weakened it. The naturally occurring isoflavones (9 and 10), which were reported to possess estrogen activity, showed moderate attracting activity.
To further the understanding of the complexity of cyclosporin A (CyA) phamacokinetics, we conducted an erythrocyte uptake and efflux study, and a protein binding study in human blood. The uptake study showed that the transport of CyA from the extracellular fraction to erythrocytes was retarded by increased human serum albumin (HSA) and lipid levels in this fraction. In addition, the concentration of CyA in erythrocytes increaesd with increases in CyA concentration in blood and reductions in hematocrit. The efflux study showed that the transport of CyA from erythrocytes to the extracellular fraction was essentially enhanced by increases of HSA and lipid levels in that fraction, but that these effects were relatively small. There were two affinity binding sites for CyA in ghost-free erythrocyte hemolysate, but not in the plasma fraction. The affinity binding constants for these binding sites were reduced by elevations in temperature, and under physiological conditions, 37°C, almost all the CyA in erythrocytes was bound to a CyA binding protein, namely, cyclophillin. These findings suggest that CyA distribution in blood is of two different types which are present in the erythrocyte and plasma fractions, respectively. Monitoring of blood biochemistry variables showed that the concentration of CyA in erythrocytes had an interlocking relationship with these physiological factors, which were related to patient disease state, i.e., hematocrit, lipids, albumin, and total protein; the concentration of CyA in erythrocytes could be predicted from these physiological factors.
Diethylstilbestrol and its derivatives were tested to determine their inhibitory effects on the relaxation activity of DNA topoisomerase I using cell lysates from Chinese hamster V79 cells. Among the derivatives investigated, (±)-indenestrol B, (±)-IB, showed the strongest inhibitory effect.
Staphylococcus aureus TPR-27, a clinically isolated strain, showed constitutive resistance to some macrolide antibiotics (erythromycin, oleandomycin, spiramycin, and josamycin), but susceptibility to the other macrolide (tylosin, rokitamycin, and mycinamicin), lincosamide, and streptogramin type B antibiotics (PM-resistance). The PM-resistant strain TPR-27 has carried for visible plasmids. Attempts to eliminate the resistant determinant in terms of ethidium bromide (about 3 μg/ml) did not succeed, and every trial to transduce the PM-resistant determinant into rec- mutant ISP105 using phage 80L2 propagated on strain TPR-27 also failed at the frequency of less than 1.1×10-10 transductants per plaque-forming unit. These results suggest that the PM-resistance determinant is localized in chromosomal DNA.
The interaction of endothelin-1 (ET-1) with either interleukin-1 β (IL-1 β) or tumor necrosis factor α (TNF α) on the release of tissue plasminogen activator antigen (t-PA : Ag) and plasminogen activator inhibitor-1 antigen (PAI-1 : Ag) was investigated in a culture system of vascular endothelial cells derived from human umbilical vein. The t-PA : Ag release was significantly decreased by either IL-1 β or TNF α; ET-1 intensified the suppressive effect of the cytokines. In contrast, PAI-1 : Ag release was significantly increased by either IL-1 β or TNF α; ET-1 significantly reduced the stimulatory effect of the cytokines. The data suggest that endothelial cell-mediated fibrinolysis may be modulated by ET-1.
We examined the effects of purified tannins and related compounds (33 species) on NADH-ubiquinone-1 oxidoreductase activity in four kinds of organism (Paracoccus denitrificans, Bacillus subtilis, Photobacterium phosphoreum, and Thermus thermophilus HB-8) and rat liver mitochondria. In addition to pentagalloylglucose, which was reported as a potent inhibitor of NADH dehydrogenases (NDH), sanguiin H-11, oolonghomobisflavan A, and polymerized procyanidin are potent inhibitors for both types of NDH (NDH-1 and NDH-2). We found that some other tannins contained in tea are also inhibitors of NDH from all organisms.
The binding of thyroxine (T4) to bovine serum albumin (BSA) has been studied in the presence and absence of Ca2+, Cu2+ and Zn2+ ions at various pH's in 0.1 M Tris-acetate buffer at 25°C using the fluoresence method. In the presence of 50 μM Ca2+ and Zn2+ and the absence of metal ions, the binding constant (K) increased similarly with increasing pH values from pH 5 to pH 9, and the K value near midpoint, pH 7.4, was 1.66±0.17×106 M-1. By contrast, the binding constant remained constant between pH 5 and pH 9 in the presence of 10 μM Cu2+, with an average value of 1.61±0.22×106 M-1, suggesting a significant influence of Cu2+ ions on T4 binding to BSA.
The effects of zonisamide (ZNS) on the pharmacokinetics of phenobarbital (PB), valproic acid (VPA), carbamazepine (CBZ) and phenytoin (PHT) were investigated in rats. Additionally, the influence of ZNS on the serum protein binding, erythrocyte distribution and metabolism of these antiepileptics was studied in vitro. The t1/2 and AUC values of PB were significantly increased by ZNS coadministration, and a significant decrease in the Vd/F value of PHT was observed after multiple dosing of ZNS. By contrast, ZNS showed no significant effect on VPA and CBZ kinetics. No significant effect of ZNS was observed in the serum protein binding, erythrocyte distribution or metabolism of other antiepileptics. These results suggest that ZNS has little effect on the pharmacokinetic behaviors of other antiepileptic drugs.
The convulsant interaction between enoxacin (ENX), a new quinolone antibacterial agent (NQ), and felbinac (FLB), a non-steroidal anti-inflammatory drug (NSAID), in vivo was reproduced as the change of GABA-induced current response in Xenopus oocytes injected with mouse brain mRNA. GABA (10 μM) response was inhibited by ENX in a dose-dependent manner, and IC50 of ENX was 96 μM. Moreover, the inhibitory effect of ENX was 80-fold potentiated in the presence of 10 μM FLB. The GABAA-antagonistic interaction between these two drugs in vitro was considered a possible mechanism of convulsant reaction after concomitant administration of NQs and NSAIDs in vivo.
Total DNA was extracted from leaves of Bupleurum falcatum originating from eight different habitats in Japan and hybridized with digoxigenin-labeled rice ribosomal DNA after digestion with various restriction enzymes. The resulting DNA fingerprints allowed us to distinguish the geographic populations of B.falcatum when the DNA was digested with Dra I, Kpn I or Taq I. A dendrogram based on the RFLP profiles indicated that B.falcatum distributing in northern Kyushu and Yamaguchi Prefecture is classified into a taxon or subtaxon that can be differentiated from the plants grown in other regions.
2-(3-Amino-3-carboxypropyl)-isoxazolin-5-one (ACl), a neurotoxic amino acid from Lathyrus odoratus, was confirmed to be derived enzymatically from S-adenosyl-L-methionine (SAM) and isoxazolin-5-one. Some properties of an enzyme in the biosynthesis of ACl are described.