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The branched-chain amino acids (BCAAs) valine, leucine and isoleucine are essential amino acids that are critical for animal growth and development. Animals need to obtain BCAAs from their diet because they cannot synthesize them. Plants are the ultimate source of these amino acids. Acetolactate synthase (ALS) is the first common enzyme in the biosynthesis of BCAAs. The metabolic control of BCAA biosynthesis involves allosteric regulation of ALS by the end-products of the pathway, i.e., valine, leucine and isoleucine. ALS holoenzyme seems to consist of two large catalytic subunits and two small regulatory subunits. In a previous study, using homologous recombination dependent gene targeting we created rice plants in which W548Land S627I mutations were induced into the endogenous gene encoding the ALS catalytic subunit. These two amino acid substitutions conferred hypertolerance to the ALS-inhibiting herbicide bispyripac-sodium. In this study, we revealed that feedback regulation by valine and leucine was reduced by these two amino acid substitutions. Furthermore, in leaves and seeds of ALS mutants with W548Land/or S627I substitution, a 2- to 3-fold increase in BCAAs was detected. Our results suggest that the ALS catalytic subunit is also involved in feedback regulation of ALS, and that judicious modification of the regulatory and catalytic subunits of ALS-coding genes by gene targeting can lead to the efficient accumulation of BCAA in plants.

Pyribencarb is a novel benzylcarbamate-type fungicide, which is active against a wide range of plant pathogenic fungi. In this paper, the inhibitory effects of this fungicide on the electron transport system of fungi, plants, rat and carp were examined to elucidate its mode of action and selectivity. Pyribencarb potently inhibited succinate-cytochrome c reductase (SCR) activities of Botrytis cinerea (cucumber gray mold), Corynespora cassiicola (leaf spot) and decylubiquinol-cytochrome c reductase (UCR) activity of B. cinerea. Pyribencarb inhibited the UCR of B. cinerea in an uncompetitive manner with respect to decylubiquinol, which was the same as strobilurin fungicides, and the substrate-dependent inhibition constant was found from calculation to be 13 nM. These results suggested that the target site of pyribencarb is cytochrome b of complex III in the electron transport system of the respiratory chain. On the other hand, the inhibitory potency of pyribencarb on SCR activities of plants, rats and carp was relatively weak compared with that of strobilurin fungicides, indicating that pyribencarb is a Qo inhibitor of cytochrome b, whose properties are superior to well-known Qo inhibitor fungicides in terms of target. The binding site of pyribencarb on cytochrome b was assumed to be a little different from that of strobilurin fungicides, because pyribencarb inhibited SCRs of strobilurin fungicide-resistant strains of B. cinerea and C. cassiicola with relatively low concentrations. The binding site was also discussed through comparison of amino acid sequences of plants, rats, carp, yeast and fungi, including B. cinerea, which was elucidated in this paper.

Previously, we showed that four mutated acetolactate synthase (ALS) genes derived from rice and Arabidopsis (W548L/S627IOsALS, S627IOsALS, W574L/S653IAtALS and S653IAtALS) confer high levels of resistance to pyrimidinylcarboxylate type ALS inhibitors (Kawai et al. 2008). Mutated ALS genes of rice were obtained from rice cells cultured in the presence of an ALS-inhibitor. The mutated ALS genes of Arabidopsis, which have the same amino acid substitutions as those of rice, have been generated artificially. Here, we demonstrate that these mutated genes function as effective selectable markers for transformation of Arabidopsis. Specifically, we studied expression of the mutated ALSs in Arabidopsis and their effect on the sensitivity of transgenic Arabidopsis plants to the ALS inhibitors. Our results show that the degree of resistance to the ALS inhibitors of transformants expressing Arabidopsis mutated ALSs was greater than those of transformants expressing rice mutated ALSs. The amino acid sequences of ALSs derived from monocotyledonous plants and those derived from dicotyledonous plants were clearly divided into two clusters in a phylogenetic tree. Based on these results, it would be preferable to use rice and Arabidopsis mutated ALS genes for generating monocotyledonous and dicotyledonous transgenic plants, respectively. Moreover, our findings are particularly useful when generating transgenic plants with a known ALS nucleotide sequence. In such cases, their own ALS gene carrying these mutations could be used as a selectable marker because amino acid residues at the point of mutation are conserved among plant species.

Herbicide-resistant turfgrass can be an efficient tool that will allow easier turf maintenance. Acetolactate synthase (ALS) is the first common enzyme in the biosynthetic pathways leading to the branched-chain amino acids, and amino acid substitutions in ALS have been known to confer resistance to ALS-inhibiting herbicides. A two-point mutated rice ALS gene [OsALS (dm)] has been shown to confer strong resistance to bispyribac-sodium (BS), an ALS-inhibiting herbicide. In this study, we introduced into turf-type tall fescue (Festuca arundinacea Schreb.) the OsALS (dm) gene by using Agrobacterium-mediated transformation for conferring herbicide resistance. Stable integration of the transgene was confirmed by Southern blot analysis. Transgenic and wild-type plants were sprayed on the leaves with herbicide containing BS; approximately half of the transgenic plants were unaffected by the treatment and showed resistance to the herbicide, whereas the wild-type plants died. ALS activity in the leaf tissue of transgenic-resistant plants incubated with BS was almost equivalent to that in wild-type plants without BS and was higher than in wild-type plants incubated with BS. These indicate that the transgenic-resistant plants actively produced OsALS (dm) protein under herbicide treatment. This is the first report of herbicide-resistant transgenic tall fescue after introduction of a mutated ALS gene.

We investigated selective culturing conditions for the production of transgenic soybeans. In this culturing system, we used the acetolactate synthase (ALS)-inhibiting herbicide-resistance gene derived from rice (Os-mALS gene) as a selectable marker gene instead of that derived from bacteria, which interfered with the cultivation and practical usage of transgenic crops. T(1) soybeans grown from one regenerated plant after selection of the ALS-targeting pyrimidinyl carboxy (PC) herbicide bispyribac-sodium (BS) exhibited herbicide resistance, and the introduction and expression of the Os-mALS gene were confirmed by genetic analysis. The selective culturing system promoted by BS herbicide, in which the Os-mALS gene was used as a selectable marker, was proved to be applicable to the production of transgenic soybeans, despite the appearance of escaped soybean plants that did not contain the Os-mALS transgene.

Strategies employed for the production of genetically modified (GM) crops are premised on (1) the avoidance of gene transfer in the field; (2) the use of genes derived from edible organisms such as plants; (3) preventing the appearance of herbicide-resistant weeds; and (4) maintaining transgenes without obstructing plant cell propagation. To this end, we developed a novel vector system for chloroplast transformation with acetolactate synthase (ALS). ALS catalyzes the first step in the biosynthesis of the branched amino acids, and its enzymatic activity is inhibited by certain classes of herbicides. We generated a series of Arabidopsis (Arabidopsis thaliana) mutated ALS (mALS) genes and introduced constructs with mALS and the aminoglycoside 3′-adenyltransferase gene (aadA) into the tobacco (Nicotiana tabacum) chloroplast genome by particle bombardment. Transplastomic plants were selected using their resistance to spectinomycin. The effects of herbicides on transplastomic mALS activity were examined by a colorimetric assay using the leaves of transplastomic plants. We found that transplastomic G121A, A122V, and P197S plants were specifically tolerant to pyrimidinylcarboxylate, imidazolinon, and sulfonylurea/pyrimidinylcarboxylate herbicides, respectively. Transplastomic plants possessing mALSs were able to grow in the presence of various herbicides, thus affirming the relationship between mALSs and the associated resistance to herbicides. Our results show that mALS genes integrated into the chloroplast genome are useful sustainable markers that function to exclude plants other than those that are GM while maintaining transplastomic crops. This investigation suggests that the resistance management of weeds in the field amid growing GM crops is possible using (1) a series of mALSs that confer specific resistance to herbicides and (2) a strategy that employs herbicide rotation.

Summary We have previously reported the production of a rice cell line tolerant to the acetolactate synthase (ALS)-inhibiting herbicide bispyribac (BS), and demonstrated that the BS-tolerant phenotype was due to a double mutation in the rice ALS gene. We further indicated that while changing either of the two amino acids (W548 L or S627I) individually resulted in a BS-tolerant phenotype, conversion of both amino acids simultaneously conferred increased tolerance to BS. As the BS-tolerant cell line had lost the ability to regenerate during two years of tissue culture selection, we attempted to introduce these two point mutations into the rice ALS gene via gene targeting (GT). Using our highly efficient Agrobacterium-mediated transformation system in rice, we were able to regenerate 66 independent GT rice plants from 1500 calli. Furthermore, two-thirds of these plants harbored the two point mutations exclusively, without any insertion of foreign DNA such as border sequences of T-DNA. The GT plants obtained in the present study are therefore equivalent to non-GM herbicide-tolerant rice plants generated by conventional breeding approaches that depend on spontaneous mutations. Surprisingly, GT rice homozygous for the modified ALS locus showed hyper-tolerance to BS when compared to BS-tolerant plants produced by a conventional transgenic system; ALS enzymatic activity in plants homozygous for the mutated ALS gene was inhibited only by extremely high concentrations of BS. These results indicate that our GT method has successfully created novel herbicide-tolerant rice plants that are superior to those produced by conventional mutation breeding protocols or transgenic technology.

Acetolactate synthase (ALS) is the first common enzyme in the biosynthetic pathway of branched-chain amino acids. Mutations of specific amino acids in ALS have been known to confer resistance to ALS-inhibiting herbicides such as sulfonylureas and pyrimidinyl carboxy (PC) herbicides. However, mutations conferring exclusive resistance to PC have not yet been reported to date. We selected PC resistant rice calli, which were derived from anther culture, using one of the PCs, bispyribac-sodium (BS), as a selection agent. Two lines of BS-resistant plants carrying a novel mutation, the 95th Glycine to Alanine (G95A), in ALS were obtained. In vitro ALS activity assay indicated that the recombinant protein of G95A-mutated ALS (ALS-G95A) conferred highly specific resistance to PC herbicides. In order to determine if the ALS-G95A gene could be used as a selection marker for rice transformation, the ALS-G95A gene was connected to ubiquitin promoter and introduced into rice. PC resistant plants containing integrated ALS-G95A gene were obtained after selection with BS as a selection agent. In conclusion, novel G95A mutated ALS gene confers highly specific resistant to PC-herbicides and can be used as a selection marker.

A two-point mutated gene of acetolactate synthase (ALS) was isolated from rice cells, which was cultured together with an ALS-inhibiting herbicide, bispyribac-sodium (BS). The mutations involved residues of tryptophan at position 548 to leucine (W548L) and serine at position 627 to isoleucine (S627I). The ALS expressed in Escherichia coli from this gene showed resistance to multiple herbicides including pyrimidinylcarboxylate (PC), sulfonylurea and imidazolinone herbicides, and showed stronger resistance to PC herbicides than to other herbicides. BS, a PC herbicide, had almost no effect on the enzyme even at 100 μM, which is an approximately 10,000-fold higher concentration than the concentration required for 50% inhibition of the wild-type. The resistance level of W548L/S627I mutating ALS to BS was stronger than the additive effect predicted from the degree of resistance of each single amino acid mutated ALS. Transformed rice cells carrying this gene and a regenerated rice plant expressed resistance to BS, suggesting that this gene is useful as a selectable marker for introducing foreign traits into rice when used with PC herbicides.

The proper use of a marker gene in a transformation process is critical for the production of transgenic plants. However, consumer concerns and regulatory requirements raise an objection to the presence of exogenous DNA in transgenic plants, especially antibiotic-resistant genes and promoters derived from viruses. One approach to overcome this problem is the elimination of marker genes from the plant genome by using several site-specific recombination systems. We propose an alternative method to solve this problem using a marker gene exclusively derived from the host plant DNA. We cloned a genomic DNA fragment containing regulatory and coding sequences of acetolactate synthase (ALS) gene from rice, and mutagenized the ALS gene into a herbicide-resistant form. After transfer of this construct to the rice genome, transgenic plants were efficiently selected with a herbicide, bispyribac-sodium salt, which inhibits the activity of wild type ALS. We also analyzed the regulatory feature of the rice ALS gene promoter with the gusA reporter gene and revealed that GUS expression was observed constitutively in aerial parts of rice seedlings and root tips. The marker system consisted exclusively of host plant DNA and enabled efficient selection in a monocot crop plant, rice. The selection system can potentially be applied to generate transgenic plants of other crop species and can be expected to be publicly acceptable.

The activity of a number of O-(4,6-dimethoxypyrimidin-2-yl)salicylic acids and their thio analogs inhibiting acetolactate synthase (ALS) preparation was measured. The effects of substituents on the salicylic-benzene ring on the inhibitory activity were analyzed quantitatively with physicochemical substituent parameters. For 6-substituted (thio)salicylic acids, the activity was shown to vary parabolically with the 'intramolecular' steric parameter (E g ). In addition, the higher steric dimension of substituents in terms of the STERIMOL width or length parameter lowered the activity. The field-inductive electron-withdrawing property of the 6-substituents in terms of the Swain-Lupton-Hansch F was favorable for the activity of salicylic acid series. In 5-substituted salicylic acids, the activity was increased by electron-donating substituents with smaller size. The relationships between ALS inhibitory and herbicidal activities were also analyzed with some weed species. Both pre- and post-emergence activities against barnyard grass, Echinochloa crus-galli, were linearly related to the ALS inhibitory activity after allowing for the hydrophobic factor that may contribute to the transport processes. Those against two broad-leaved weed species, Polygonum convolvulus and Abutilon theophrasti were linearly related to the in-vitro activity with no significant participation of the hydrophobic factor.

Target-site and non-target-site herbicide tolerance are caused by the prevention of herbicide binding to the target enzyme and the reduction to a nonlethal dose of herbicide reaching the target enzyme, respectively. There is little information on the molecular mechanisms involved in non-target-site herbicide tolerance, although it poses the greater threat in the evolution of herbicide-resistant weeds and could potentially be useful for the production of herbicide-tolerant crops because it is often involved in tolerance to multiherbicides. Bispyribac sodium (BS) is an herbicide that inhibits the activity of acetolactate synthase. Rice (Oryza sativa) of the indica variety show BS tolerance, while japonica rice varieties are BS sensitive. Map-based cloning and complementation tests revealed that a novel cytochrome P450 monooxygenase, CYP72A31, is involved in BS tolerance. Interestingly, BS tolerance was correlated with CYP72A31 messenger RNA levels in transgenic plants of rice and Arabidopsis (Arabidopsis thaliana). Moreover, Arabidopsis overexpressing CYP72A31 showed tolerance to bensulfuron-methyl (BSM), which belongs to a different class of acetolactate synthase-inhibiting herbicides, suggesting that CYP72A31 can metabolize BS and BSM to a compound with reduced phytotoxicity. On the other hand, we showed that the cytochrome P450 monooxygenase CYP81A6, which has been reported to confer BSM tolerance, is barely involved, if at all, in BS tolerance, suggesting that the CYP72A31 enzyme has different herbicide specificities compared with CYP81A6. Thus, the CYP72A31 gene is a potentially useful genetic resource in the fields of weed control, herbicide development, and molecular breeding in a broad range of crop species.

Three feline coronavirus (FCoV) isolates KUK-H, M91-266, and M91-267 were examined to elucidate their biological and antigenic properties as well as disease potential in cats. Immune stainings of virus-infected cells by using FCoV type-specific monoclonal antibodies indicated that their antigenic specificity was serotype II. However, antigenic variations among these serotype II FCoVs were detected by neutralization assay with hyperimmune antisera against FCoVs and canine coronaviruses, and with experimentally infected cat sera; there were two subtypes in serotype II FCoVs. The isolates efficiently grew in fcwf-4 cell culture showing lytic CPE enough to form distinct plaques: when measured 48 hr after infection, plaque sizes of both M91-266 and M91-267 were approximately 1 mm in diameter, and a mixture of small (less than 1 mm in diameter) and large (approximately 3 mm in diameter) plaques were produced in the case of KUK-H. Strains KUK-H, M91-266 and M91-267 produced feline infectious peritonitis (FIP) in 50%, 67% and 89% of experimentally inoculated kittens, respectively. Furthermore, 80% of the kittens inoculated with the small plaque former of KUK-H developed FIP accompanied by more prominent clinical signs as well as pathological changes when compared with 28.6% of kittens inoculated with the large plaque former. These results suggest that serotype II FIPVs producing smaller size of plaques are more virulent than those producing larger size of plaques.

Acetolactate synthase (ALS) is an enzyme in the biosynthetic pathway for branched-chain amino acids, and bispyribac-sodium (BS), a pyrimidinyl carboxy herbicide, is a well-known inhibitor of ALS activity. However, it appears that a mutated form of rice ALS [OsmALS (W548L/S627I)] confers resistance to BS. We succeeded in using OsmALS with native OsALS promoter and terminator region for a selection marker of rice transformation. Because this selection marker cassette is originally from the rice endogenous genome, it can be expected to be publicly acceptable.

The inhibition of acetolactate synthase (ALS) from ethiolated pea seedlings by 2-(4,6-dimethoxypyrimidine-2-yloxy)benzoic acid (compound 2), one of the pyrimidinylsalicylic acid (PS) compounds, was desensitized by SH inhibitors but not at the alkaline pH. PS compounds including pyrithiobac (2-chloro-6-(4,6 dimethoxypyrimidine-2-ylthio)benzoic acid) tested in this study inhibited ALS activity in the mixed-type manner with respect to pyruvate and in the non-competitive with respect to thiamine pyrophosphate (TPP). These compounds inhibited both of the two molecular species of ALS which were partially purified from pea seedlings and exhibited slow-binding properties to the crude preparation of ALS from pea seedlings in the extended-time-course experiments. The inhibition constants of the intitial inhibition by PS compounds were 13- to 26-fold larger than those of the final steasy state and the maximal first order rate constant (0.69 min −1 ) for transition from the initial to the final steady state of the inhibition of pyrithiobac was nearly identical to those reported on other widely achnowledged ALS inhibitors. From these results, we conclude: PS compounds are slow-binding inhibitors to ALS activity, categorized in the sulfonylurea type. The binding site of PS compounds on the enzyme is considered to be located on the similar site to sulfonylureas. This binding site is judged not to be located on the regularoty center(s) but on the allosteric site in a wide sense near the catalytic center, which overlaps partially with the binding site of pyruvate and does not overlap with the binding site of TPP

エンドウ黄化幼植物茎葉からアセト乳酸シンターゼ (ALS) を精製したところ, 分子量の相違する2種のALSが検出されたが, 酵素的性質および精製中の挙動から判断して, 分子量の小さな分子種は大きな分子種から制御中心がはずれて生成したものと結論された. したがって, エンドウのALSは一種でありアイソザイムは存在しないものと考えられる. このALSはこれまでに報告されていない次のような性質を示した. (1) 基質である2分子のピルビン酸の間には協同性が認められない. (2) 分岐鎖アミノ酸によるフィードバック阻害は負の協同性を示す. (3) バリンによる阻害とイソロイシンによる阻害は拮抗的である. (4) フィードバック阻害の阻害形式はピルビン酸濃度に依存して変化する. (5) 分岐鎖アミノ酸によるフィードバック阻害はSH基阻害剤やATPにより脱感作されない.

棉用除草剤 pyrithiobac sodium (商品名 Staple) に代表されるピリミジニルサリチル酸系除草剤のイネ生育抑制およびクロレラ増殖阻害は, 3種の分岐鎖アミノ酸の同時添加によりほぼ完全に回復した. 本系統化合物は低濃度で分岐鎖アミノ酸生合成経路上のアセト乳酸シンターゼ (ALS) を阻害したが同経路上のケトール酸レダクトイソメラーゼには影響を与えなかった. この結果, 本系統化合物の作用点はALSであると判断定れる. 一方, pyrithiobac は棉ALSとその他の植物ALSを同程度の強定で阻害したことから, 本剤の棉に対する選択性はALSの感受性以外の要因に依存していることが示唆定れた. また, 本系統化合物は単離葉緑体の光合成電子伝達系には影響を与えなかったが, 黄化葉の葉緑素生合成には弱い阻害効果を示した.

Fifty-four canine cutaneous mast cell tumors were evaluated immunohistochemically for the expression of P-glycoprotein (PGP) and multidrug-resistance-associated protein (MRP). All tumors examined were graded according to the histological malignancy. ranging from grade I to III. The expression of PGP was confirmed in 15% (8/54) of whole, 33% (5/15) of grade I, 10% (3/31) of grade II, and 0% (0/8) of grade III tumors. The expression of MRP was found in 18% (10/54) of whole, 26% (4/15) of grade I, 19% (6/31) of grade II, and 0% (0/8) of grade III tumors. The cases positive to at least one of these 2 multidrug markers were 26%, 47%, 23% and 0% of whole and grade I to III tumors, respectively. These results indicate that at least 26% of canine cutaneous mast cell tumors express PGP and/or MRP and that these tumors may be resistant to several anti-cancer drugs.

Transgenic crops such as maize (Zea mays L.), soybeans (Glycine max L. Merr.), canola (Brassica napus L.) and cotton (Gossypium hirsutum L.) have been widely used in the field. In 2009, transgenic crops were cultivated in approximately 134 million hectares in 25 countries, mainly USA, Brazil, Argentina, India, Canada and China (http://www.isaaa.org). The adoption of transgenic crops has steadily increased since 1996 because of their many benefits for farmers. Herbicide resistance is one of the most important agronomic traits conferred onto transgenic crops. Herbicide-resistant crops comprise 62 percent of all transgenic crops (http://www.isaaa.org), and are produced by the introduction of herbicide-resistant genes using genetic transformation. Herbicide resistance can be used as an efficient tool to allow easier weed management. It facilitates control of weed species and contributes to reducing costs, labor, and the waste of chemical spray. Herbicide resistance can also facilitate the selection of transgenic cells from non-transgenic cells as a selectable marker in genetic transformation. Acetolactate synthase (ALS)-inhibiting herbicides are widely used around the world. ALSinhibiting herbicide-resistant weeds were first found in kochia (Kochia scoparia L. Shrad) (Primiani et al., 1990) and prickly lettuce (Lactuca serriola L.) (Mallory-Smith et al., 1990). Subsequently, plants and cultured cells resistant to ALS-inhibiting herbicides have been generated using both conventional mutation breeding and somatic cell selection. Since then, the ALS genes have been cloned and characterized. In most cases, resistance to ALSinhibiting herbicides has been found to be conferred by single or double amino-acid mutations at a particular position in ALS. Mutated ALS genes can be used not only for the generation of herbicide-resistant crops, but also as selectable markers. We are now producing transgenic tall fescue (Festuca arundinacea Schreb.) and maize that are resistant to ALS-inhibiting herbicides using novel mutated ALS genes. This chapter focuses on mutated ALS genes and their application to the production of herbicide-resistant crops and selection of transgenic cells as selectable markers.

Bispyribac sodium (BS), a pyrimidinyl carboxy herbicide, is a well-known inhibitor of acetolactate synthase (ALS) activity. ALS is an enzyme in the biosynthetic pathway for branched-chain amino acids. A mutant form of rice ALS (OsmALS [W548L/S627I]) that confers resistance to BS can be used as an in vitro selection marker gene for plant transformation. Since indica and indica-derived cultivars are thought to have lower BS sensitivity than japonica rice, the application of BS as a selectable reagent for genetic transformation in indica and indica-derived cultivars is more challenging than for japonica cultivars. In this study, callus and seedlings of eight different rice cultivars (five indica-derived cultivars, two indica cultivars and one japonica cultivar) were tested for BS sensitivity. Our study indicates for the first time that callus shows a higher sensitivity to BS than seedlings in indica and indica-derived cultivars. We used BS with OsmALS [W548L/S627I] to select transformed calli, and transgenic rice plants from indica and indica-derived cultivars were successfully obtained.

Acetolactate synthase (ALS; EC 4.6.3.8, also referred to acetohydroxy acid synthase; AHAS) is the first common enzyme in the biosynthetic pathway to the branched-chain amino acids; valine, leucine and isoleucine (Fig. 1). The pathway exists in plants and microorganisms such as bacteria, fungi and algae. ALS is the primary target site of action for at least four structurally distinct classes of herbicides including the sulfonylureas (SUs; LaRossa and Schloss 1984; Ray 1984), the imidazolinones (IMs; Shaner et al. 1984), the triazolopyrimidine sulfonamides (TPs; Subramanian and Gerwick 1989) and the pyrimidinylsalicylates (pyrimidinyl carboxy herbicides, PCs; Shimizu et al. 1994b), all of which have been successful in their development as commercial herbicides. The extremely good weed control activity achieved with these herbicides indicates that ALS is a very effective target site for herbicidal action. Deficiency of the pathway of branched-chain amino acids biosynthesis in mammals (Singh and Shaner 1995) shows us that it is the selective target between plants and mammals. ALS is therefore attractive for addressing a large number of goals of modern herbicide research.

Two of the 4 goats given a daily dose of 4-6mg/kg cycasin for 88 to 124 days presented weakness and paretic signs in the hindquarters. All the 4 goats showed anorexia, weight loss, anemia and decreases in serum albumin and γ-glutamyl transpeptidase (γ-GTP) levels. Two of them had severer hepatic lesions and leukomyelopathy consisting of demyelination and axonal swelling or disappearance in the spinal cord, which were similar to those seen in cycad poisoning of cattle.