A lectin was isolated from hemolymph of the Southeast Asian horseshoe crab Tachypleus gigas by using glycophorin HA affinity chromatography and Sephacryl S-300 gel filtration. The purified lectin had a molecular mass of approximately 396kDa and was composed of 13 identical subunits with molecular masses of 31kDa. The serological specificity of the purified lectin was specifically inhibited by sialic acids sialoglycoproteins, but not by neutral sugars, hexosamines, N-acetylhexosamines, or asialoglycoproteins. Although the N-terminal amino acid sequence of the lectin from T. gigas was identical to that from American horseshoe crab (liphemin) by the same purification method and cross reacted with the anti-liphemin serum, the calcium concentration of hemagglutinating activity of the purified lectin showed a smaller optimal concentration than that of liphemin.

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The proteinaceous α-amylase inhibitor, T-76, gene was cloned by screening a Streptomyces nitrosporeus genomic library using a deoxyinosine-containing probe corresponding to the amino acid sequence of the inhibitor. The nucleotide sequence of the insert of a positive clone had an open reading frame of 330bp that encoded a polypeptide of 110 amino acid residues with a calculated molecular mass of 11, 306 daltons. The polypeptide begins with proximal basic amino acids and a region rich in hydrophobic amino acids that possibly act as a signal peptide for secretion, which is followed by a sequence consistent with the amino-terminal amino acid sequence of the T-76 inhibitor. Escherichia coli cells harboring the plasmid derivatives for expression produced the inhibitor in their periplasmic space. The amino-terminal sequence of the inhibitor produced by an E. coli transformant was identical to that of the T-76 inhibitor secreted by S. nitrosporeus. The amino acid sequence of the inhibitor deduced from nucleotide sequence showed significant homology to other proteinaceous α-amylase inhibitors.

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Human erythropoietin (Epo) cDNA was engineered for expression in cultured tobacco cells (Nicotiana tabacum L. cv. BY2). Two plasmid DNAs were constructed : pCEP, which contained Epo cDNA under control of the cauliflower mosaic virus-derived 35S RNA promoter and terminator, and pNSEP, which contained signal sequence-deleted Epo cDNA under control of the 35S RNA promoter and terminator. By using the electroporation method, each of these plasmid DNAs was transferred into the protoplasts of BY2 cells together with a plasmid, pNR35, which conferred G418-resistance on the cells. Four G418-resistant clones were obtained from protoplasts transfected with pNSEP and pNR35, and only one of them, named 11N ; survived in suspension culture. Integration of pNSEP DNA into the genome of 11N cells was confirmed by Southern blot and PCR analyses. Production of Epo mRNA was shown by Northern blot analysis. Epo protein was shown to be expressed in 11N cells by colorimetric enzyme immunoassay. The productivity of Epo in the 11N cells (1 pg/g of wet cells) was very low.

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Four tuber-forming substances in Jerusalem artichoke were isolated from the leaves. The structures were established by spectroscopic methods as jasmonic acid (2), methyl β-D-glucopyranosyl tuberonate (3), and two new Polyacetylene compounds, methyl β-D-glucopyranosyl helianthenate A (4, C₁₉H₂₄O₈) and B (5, C₁₇H₂₂O₈).

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Rheological and structural properties of legumin gels, treated with various reagents, were studied. The gels were hardened by propylene glycol and ethanol, but softened by 2-mercaptoethanol, NaSCN, and formamide. The gel was completely dissolved by 8 M urea, but soluble aggregates, structural units of the gel network, remained. A combination of NaSCN with either 2-ME or formamide did not dissolve gels completely, but significantly decreased gel hardness. Treatments with 2-ME and urea decreased elastic parameters and viscous parameters, respectively. These results indicated that disulfide bonds play a role in stabilization and promote the elasticity of legumin networks, while hydrogen bonds and hydrophobic interactions predominate in maintaining the structure and increasing the viscosity.

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Amidenin, a new plant growth-regulating substance, was isolated from the fermentation broth of an Amycolatopsis sp. The active principle was extracted with ethyl acetate and purified by silica gel column chromatography. The molecular formula of this substance was established as C₁₀H₁₇NO, and the structure was determined to be 2E, 4Z-decadienamide by spectroscopic analyses. This substance showed growth-promoting activity with a 0.6×10^-5-1.8×10^-5M treatment to rice seedlings, and inhibitory activity with a 6.0×10^-5M treatment.

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The survival of wild-type and genetically engineered Pseudomonas putida PpY101 that contained a recombinant plasmid pSR134 conferring mercury resistance were monitored in aquatic microcosms. We used lake, river, and spring water samples. The density of genetically engineered and wild-type P. putida decreased rapidly within 5 days (population change rate k -0.87∼-1.00 day^-1), then moderately after 5 to 28 days (-0.10∼-0.14 day^-1). The population change rates of genetically engineered and wild-type P. putida were not significantly different. We studied the important factors affecting the survival of genetically engineered and wild-type P. putida introduced in aquatic microcosms. Visible light exerted an adverse effect on the survival of the two strains. The densities of genetically engineered and wild-type P. putida were almost constant until 7 days after inoculation in natural water filtered with a 0.45-μm membrane filter, or treated with cycloheximide to inhibit the growth of protozoa. These results suggested that protozoan predation was one of the most important factors for the survival of two strains. We examined the impact of the addition of genetically engineered and wild-type P. putida on indigenous bacteria and protozoa. Inoculation of genetically engineered or wild-type P. putida had no apparent effect on the density of indigenous bacteria. The density of protozoa increased in microcosms inoculated with genetically engineered or wild-type P. putida at 3 days after inoculation, but after 5 to 21 days, the density of protozoa decreased to the same level as the control microcosms.

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The fruit body formation of Schizophyllum commune was greatly stimulated by the addition of papulacandin B, aculeacin A, gramicidin S, luteinizing hormone-releasing hormone (LH-RH), or digitonin to the culture media. Papulacandin B and aculeacin A inhibit glucan synthase, which was demonstrated with S. commune in vitro in this study. The in vitro S. commune glucan synthase was activated by phospholipids that counteracted the stimulation of fruit body formation by papulacandin B. LH-RH, however, did not inhibit the glucan synthase. Digitonin was known to inhibit chitin synthase, but polyoxin B, a substrate analogue inhibitor of chitin synthase, did not stimulate the fruit body formation. The characteristic common to most of these stimulators of fruit body formation is possible induction of structural changes of plasma membranes to which the substances can attach with their biphasic nature. These stimulators of fruit body formation were mostly accompanied by an evident suppression of hyphal growth, independent of phenoloxidase activity, and not observed in continuous dark or with monokaryotic strains.

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One strain (MO-5) of fungi producing α-D-xylosidase was isolated from sake koji by a method using glucose oxidase. MO-5 was identified as an Aspergillus flavus strain, produced no aflatoxin (B1, B2, G1, and G2). Two different types of α-D-xylosidases were detected in the cell-free extract. One of the enzymes (α-D-xylosidase I) was purified to an electrophoretically pure state by successive chromatography on Q-Sepharose, Phenyl Superose, PL-SAX, and TSK-gel G3000SW_XL. The purified enzyme hydrolyzed p-nitrophenyl α-D-xylopyranoside (α-p-NPX), methyl α-D-xylopyranoside, isoprimeverose [α-D-xylopy-ranosyl-(1→6)-D-glucopyranose], and xyloglucan oligosaccharide. The activity of this enzyme was highly specific for α-D-xylosidic linkages. The apparent K_m and V_max of the enzyme for α-p-NPX and isoprimeverose were 1.32 mM and 4.4 μmol/min/mg protein, and 4.00 mM and 58.8 μmol/min/mg protein, respectively. This enzyme had an apparent molecular weight of 100, 000 by SDS-polyacrylamide gel electrophoresis and 400, 000 by gel filtration chromatography (TSK-gel G3000SW_XL). The enzyme showed the highest activity at pH 4.5 and 45°C, and was stable from pH 4.5 to 6.0 and at temperatures up to 45°C. The activity was inhibited by SDS and Hg²⁺, and slightly by Cu²⁺ and Fe³⁺. This enzyme showed transfer action at high concentrations of isoprimeverose and transfer products were detected, and it had transxylosylation activity on maltose from isoprimeverose as a donor, too. From the results of the enzymatic digestion and the partial acidolysis of these prepared transfer products, their structures may be trisaccharides in which the xylosyl residues were transferred to isoprimeverose and maltose forming α-D-linkages. Accordingly, this enzyme was indicated to be able to transfer xylosyl residues to form α-D-xylosidic linkages.

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α-D-Xylosidase II activity from Aspergillus flavus MO-5 was increased roughly 5- to 10-fold by use of xylose instead of methyl α-D-xylopyranoside (α-MX) as a carbon source. The enzyme was purified to an electrophoretically pure state by successive chromatography on Q-Sepharose, Phenyl Superose, PL-SAX, and TSK-gel G3000SW_XL. The purified enzyme hydrolyzed isoprimeverose [α-D-xylopyranosyl-(1→6)-D-glucopyranose] and p-nitrophenyl α-D-xylopyranoside (α-p-NPX), but not α-MX or xyloglucan oligosaccharide. The apparent K_m and V_max of the enzyme for α-p-NPX and isoprimeverose were 0.97 mM and 28.0 μmol/min/mg protein, and 47.62 mM and 2.0 μmol/min/mg protein, respectively. This enzyme had an apparent molecular weight of 67, 000 by SDS-polyacrylamide gel electrophoresis and 180, 000 by gel filtration chromatography (TSK-gel G3000SW_XL). The enzyme showed the highest activity at pH 6.0 and 40°C, and was stable in the pH range from 6.0 to 7.0 and at the temperatures up to 40°C. The activity was inhibited by Cu²⁺, Zn²⁺, Hg²⁺, p-CMB, SDS, Fe³⁺, and N-ethylmaleimide. This enzyme had nothing in common with α-D-xylosidase I and four α-D-xylosidases reported already.

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Production of theobromine from caffeine by caffeine-degrading bacteria was studied. We found that addition of metal ions such as Zn²⁺ to intact cells of a caffeine-degrading isolate from soil, Pseudomonas sp. No. 6, resulted in a high theobromine accumulation from caffeine. We hypothesized that Zn²⁺ acts as a selective inhibitor of one of the theobromine-demethylating enzymes and further screened for theobromine-producing activities in the presence of Zn²⁺ among a number of caffeine-using microorganisms. A strain identified taxonomically as Pseudomonas putida No. 352 showed the best productivity among 973 microorganisms of stock cultures and soil isolates. Culture conditions for the production of theobromine from caffeine by P. putida No. 352 were studied. Under optimal conditions, nearly 20 g/liter of theobromine was produced from caffeine in a yield of 92%.

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Vanillin was converted into the corresponding glucoside in suspension-cultured cells of Ceffea arabica. The maximum efficiency of glucosylation was 85% within 24 h after the addition of 1 mM vanillin when cultured in a modified Murashige and Skoog's medium with 5 μM 2, 4-dichlorophenoxyacetic acid and 0.5 μM kinetin. The glucoside was identified as 4-formyl-2-methoxyphenyl-O-β-D-glucopyranoside by ¹H-NMR, ¹³C-NMR, FAB-MS, and hydrolysis by α- and β-glucosidases. It retained the antimutagenic and antimicrobial activities of vanillin.

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In repeated glycosylmoranolines synthetic reaction at 55°C, cyclodextrin glycosyltransferase (CGT-ase, EC 2.4.1.19) from Bacillus stearothermophilus retained its activity for more than 600 days. A main stabilizing compound was found to be 4-O-α-D-glucopyranosylmoranoline. The thermostabilizing activities of moranoline, 4-O-α-D-glucopyranosylmoranoline, and their N-substituted derivatives were studied. Moranoline and its N-substituted derivatives stabilized glucoamylase. 4-O-α-D-Glucopyranosylmoranoline and its N-substituted derivatives stabilized CGT-ase and β-amylase.

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A novel salicylaldehyde analog was isolated in a 3.7% yield from the hexane extract of the acarid mite Dermatophagoides pteronyssinus, possibly as its opisthonotal gland secretion. The chemical structure was physico-chemically elucidated to be 2-formyl-3-hydroxybenzyl formate, to which we give the trivial name rhizoglyphinyl formate, and the identity of the compound was confirmed by its synthesis.

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Various phosphatidylsaccharides can be produced in good yields by a one-step transphosphatidylation reaction catalyzed by the purified phospholipase D from Actinomadura sp. strain No. 362. With glucose as the saccharide, the optimal conditions of transphosphatidylation were investigated. Several solvents such as diethylether or tert-butanol and several salts such as NaCl or CaC1₂ were effective for phosphatidylglucose production. The optimal concentration of glucose for phosphatidylglucose production was 0.8 part of the water. With optimal conditions, the conversion rate of egg lecithin to phosphatidylglucose reached about 85%. With this condition of phosphatidylglucose production, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, soya lecithin, phosphatidylethanolamine, phosphatidylglycerol, dihexadecylphosphatidylcholine, and sphingomyelin were found to be good substrates. Pentoses, hexoses, and heptoses with primary alcohol groups were also found to be good acceptors of transphosphatidylation.

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Rapid-turnover plasma proteins (transferrin, transthyretin, and retinol-binding protein) have been proposed to be sensitive nutritional parameters. In this study, we investigated the daily changes in the rapid-turnover protein levels of rats whose nutritional conditions were controlled by parenteral nutrition under the acute-phase response. We measured the plasma fibrinogen level to monitor the acute-phase response, and the level shows that the acute-phase response was induced by the surgical treatment and ended on day 3. The plasma transferrin level in TPN solution-infused rats increased to more than the pre-infusion level from day 1, and was significantly higher than that in saline solution-infused rats on and after day 3. The plasma transthyretin and retinol-binding protein levels of the TPN group were significantly higher than those of the saline group on and after days 2 and 3, respectively. These results indicate that rapid-turnover proteins are useful for estimating the nutritional status in injured rats under parenteral nutrition.

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By controlling the concentrations of kinetin, auxin, and sucrose in the Murashige-Skoog medium, starch contents in callus culture induced from sweet potato tissues could be manipulated. Activity staining and Western analysis on PAGE plates and activity assays made on starch phosphorylase in the presence and absence of mercuric ions showed that β-amylase is absent in callus cultures regardless of whether their starch content is high or low. This would imply that β-amylase induction in sweet potato calli is not linked to the metabolic control through which the expression of storage function is associated, as proposed by Nakamura et al. [Plant Physiol., 96, 902 (1991)] for sweet potato leaf-petiole cuttings. Analyses of starch phosphorylase in crude extracts suggested the presence of a new starch phosphorylase in tuberous root and callus tissue. This phosphorylase is immunologically different from the tuberous root and leaf enzymes that we studied previously.

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Endo-1, 4-β-D-mannanase (1, 4-β-D-mannanohydrolase, EC 3.2.1.78) was purified from viscera of a mud snail, Pomacea insularus (de Ordigny). The purified enzyme gave a single protein band in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the purified enzyme was estimated to be 44, 000. The amino-terminal sequence was H·Gly-X-Leu-Arg-Arg-Gln-Gly-Thr-Asn-Ile-Val-Asp-Ser-His-Gly-His-Lys-Val-Phe-Leu-Ser-Gly-Ala-Asn-Thr-Ala-Trp-Val-Ala-Tyr-Gly-Tyr-Asp-. The enzyme was stable from pH about 5.0 to about 10.5 and had its maximum activity at pH about 5.5. The purified enzyme produced M2, M3, M4, and M5 from β-1, 4-mannan. Enzyme activity was greatly inhibited by Ag⁺, Hg²⁺, Cu²⁺, and dithiothreitol at 1 mM concentration. In addition, N-bromosuccinimide completely inhibited the enzyme activity.

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Among the yeasts belonging to the genus Torulaspora, T. pretoriensis IFO 10218 was selected as a maltose-fermenting strain that could leaven dough containing 5% glucose as high as commercial baking strains. The freeze-thaw resistance of this strain in the dough was higher than that of Saccharomyces cerevisiae FL 2209 isolated from commercial compressed yeast. Since the cells of IFO 10218 readily sedimented in their suspension, a dispersing mutant, YK-1, was induced. When YK-1 was cultured in the presence of NaCl, leavening abilities were reduced, unlike FL 2209. α-Glucosidase activity of YK-1 cells grown on maltose increased slightly, resulting in partly improved leavening ability in the dough without addition of sugar. YK-1 was shown to be a candidate applicable to breadmaking by not only usual method but also the frozen-dough method.

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Southern hybridization analysis using the genes encoding the α- and β-subunits of nitrile hydratase (NHase) from Rhodococcus sp. N-774 as probe suggested that two R. erythropolis strains, JCM6823 and JCM2892, among 31 strains mainly from Japan Culture of Microorganisms (JCM) have NHase genes. Restriction analysis of DNA fragments showing positive hybridization showed that each fragment carried a nucleotide sequence very similar to that of the NHase genes from Rhodococcus sp. N-774. Nucleotide sequence analysis of the DNA fragment cloned from R. erythropolis JCM6823 showed the presence of the genes encoding the α- and β-subunits of NHase, which show 94.7% and 96.2% identity in amino acid sequence to those of Rhodococcus sp. N-774, respectively, as well as a C-terminal portion of the amidase gene upstream from these genes. Despite the extremely high amino acid sequence similarity in both NHases and amidases from R. erythropolis JCM6823 and Rhodococcus sp. N-774, the NHases and amidases from R. erythropolis strains showed broader substrate specificity when compared to those from Rhodococcus sp. N-774. This suggests that a very limited number of amino acid residues are responsible for the difference in substrate specificity. Although the NHase of Rhodococcus sp. N-774 are constitutively produced, the NHases of both R. erythropolis strains were inducibly produced by addition of ε-caprolactam as an inducer.

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Amides in a CH₂Cl₂ extract from the fruits of Piper retrofractum were detected by HPLC/APCI-MS. Seven new unsaturated amides, together with six known ones, were isolated, and their structures were determined to be N-isobutyl-2E, 4E, 12Z-octadecatrienamide (1), N-isobutyl-2E, 4E, 14Z-eicosatrienamide (2), 1-(octadeca-2E, 4E, 12Z-trienoyl)piperidine (3), 1-(eicosa-2E, 4E, 14Z-trienoyl)piperidine (4), 1-(octadeca-2E, 4E-dienoyl)piperidine (5), 1-(eicosa-2E, 4E-dienoyl)piperidine (6), and 1-(eicosa-2E, 14Z- dienoyl)piperidine (7) on the basis of chemical and spectroscopic evidence.

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We screened microorganisms for those that resolved a stereoisomer from rac-1-phenoxy-2-propyl acetate (PPAc). Several species of Nocardia and Rhodococcus hydrolyzed rac-PPAc to rac-1-phenoxy-2-propanol (PPol), and then selectively decomposed an isomer of PPol, leading to an accumulation of (R)-PPol. Several yeasts and bacteria hydrolyzed PPAc asymmetrically, affording (R)-PPol and the ester of (S)-PPol. An enzyme that catalyzed the asymmetric hydrolysis of PPAc was partially purified from Corynebacterium glutamicum ATCC 13059 and characterized. The enzymatic hydrolysis was highly specific for (R)-PPAc.

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The molecular conformation of elsinan, consisting of (1→3)-α-linked maltotriose and α-maltotetraose units, was studied by X-ray diffraction coupled with conformational analysis. The quality of the X-ray fiber pattern obtained from elsinan was very poor, but the layer line spacing (45Å), the probable presence of (005) reflection and a similar pattern with the powder pattern of a low molecular weight poly[(1→3)-α-maltotriose] segment (DP about 35) suggested that the poly[(1→3)-α-linked-α-maltotriose] segment (MTR part) of elsinan chain took a five-fold helical structure with an asymmetric unit of maltotriose. Conformational analysis for the five-fold helix of the MTR part pointed out that two left handed helices, -5/1 and -5/2, were energetically probable.

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Plasminogen activator (PA) produced by rat hepatocytes (hepatic PA, h-PA) was purified from 5.2 liters of the conditioned medium of primary cultures. The three-step procedure, consisting of a carboxymethyl-Sepharose gel, arginine-Sepharose column, and benzamidine-Sepharose column chromatographies yielded 35μg of h-PA with a specific activity of 42, 800IU/mg. The molecular mass of the purified h-PA was 70kD, which is similar to that of rat bilokinase (BK), a biliary PA, 70kD ; and to that of human melanoma tissue-type PA (t-PA), 72kD. However, it is quite different from that of high (54 kD) or low (31 kD) molecular weight urinary PA, urokinase (UK). The h-PA had a much higher affinity for fibrin than UK. Unlike UK activity, the h-PA activity was augmented by CNBr-digested fibrinogen fragments (a stimulator). Thus h-PA should be categorized as a tissue-type PA, and may have high efficacy for recanalization of vessels occluded by thrombi. The characteristics of h-PA are almost identical to those of BK, which we reported previously [J. Biol. Chem., 258, 622-28 (1983)].

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A bacterium capable of using collagen as sole carbon and nitrogen sources was isolated and identified as Bacillus alvei DC-1. The specific activity of the ammonium sulfate fraction (80% saturation) was 3.81×10⁷ Mandle units/mg. Also, in the collagen substrate, the optimum pHs were 4.5, 6.0, and 7.0. This strain produced a new type of collagenase, which had optimal activity at an acidic pH.

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The solubility of ferric iron with lactoferrin (Lf) was investigated in the presence of bicarbonate and phosphate salts. Lf and Lf digested with pepsin and trypsin could solubilize over a 70-fold molar equivalent of iron, which is much higher than the specific iron-binding ability of Lf. Lf appears promising as an ingredient for dietetic foods as it protects against iron-deficient anemia.

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The rate parameters for maltooligosaccharides of α-glucosidase from Mucor javanicus were studied. The K_m and k_o values (mM, s^-1) for maltose, malto-triose, -tetraose, -pentaose, -hexaose, and -heptaose were estimated to be (0.60, 334); (0.29, 271); (0.27, 312); (0.23, 285); (0.14, 230); (0.11, 184) in this order. The subsite affinities in the active site were 1.78, 5.31, 0.317, 0.126, 0.039, 0.168, and 0.002 kcal/mol for subsites 1, 2, 3, 4, 5, 6, and 7, respectively. From these results, it is considered that extended subsites exist in the enzyme, where maltooligosaccharides could be bound.

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Benzyl alcohol oxidase (BAO) has been assayed for the culture medium and mycelia of Botrytis cinerea, grown in the presence of aromatic alcohols with either glucose or galactose. Veratryl and coniferyl alcohols increased the BAO activities of both fraction. Activities were highest with the combination of veratryl alcohol and galactose. The implication of BAO in the degradation of lignin-related compounds with regards to the host-parasite interaction is discussed.

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Degradation product including the moiety for C-19 through to C-25 of tautomycin were synthesized. This synthesis confirmed the relative stereochemistry at C-23 and C-ν of tautomycin.

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To investigate the role of signal peptides in extracellular production in Bacillus brevis, the Bacillus stearothermophilus α-amylase signal peptide was systematically altered and the effects were analyzed in B. brevis. Efficient extracellular production of the amylase in B. brevis was accomplished either by introducing point mutations at positions between -6 and -4 or by replacing the whole signal peptide with that of B. brevis middle wall protein.

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7-(4-Hydroxyphenylacetamido)cephalosporanic acid (1) was transformed into 7-[1-oxaspiro(2.5)octa-6-oxo-4, 7-diene-2-carbox-amido]cephalosporanic acid (2) by laccase-catalyzed phenolic oxidation. 2 consisted of two diastereomers, named CXL-1 and CXL-2. CXL-2 was 10-30 times more active than CXL-1, although less active than 1.

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The photosystem II inhibitory activity of substituted phenylurea derivatives in liverwort photoautotrophically cultured cells was determined by monitoring chlorophyll fluorescence. Since the inhibitory activities in this assay correlated well with both the Hill inhibitory activity in isolated thylakoids and the herbicidal activity against mung bean plants, this bioassay may be a suitable screening method for photosystem II inhibitory herbicides.

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Saccharomyces cerevisiae strains carrying the mutant allele skt5 are resistant to the killer toxin of Kluyveromyces lactis and are defective in protoplast regeneration. The DNA sequence analysis of the cloned mutant skt5 gene showed a nucleotide substitution (causing a glycine-to-glutamic acid substitution) and also a nucleotide insertion (causing a frameshift at the extreme carboxy-terminal region) in the structural region from its wild-type gene.

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(+)-(1R, 2S)-Allocoronamic acid, (-)-(1S, 2R)-allocoronamic acid, and their racemate showed an inhibitory effect on senesence in cut carnation flowers. This antisenescent activity was based on the inhibition of endogenous ethylene formation catalyzed by an ethylene-forming enzyme.

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Chitinase (Chi85) from Alteromonas sp. strain O-7 contains the two conserved regions common to microbial and plant chitinases. We did site-directed mutagenesis of Chi85 to investigate the effects of the conserved amino acid residues on chitinase activity. We suggest that Asp-290 and Glu-292 of Chi85 may be the essential amino acid residues for the cleavage of β-glycosidic linkage of chitin.

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A good scopolamine-releasing hairy root clone, DL47-1, was developed from Duboisia leichhardtii. Hyoscyamine 6β-hydroxylase activity of the clone DL47-1 in the medium for alkaloid release was 2-fold higher than that in the medium for hairy root growth. Other high scopolamine-producing clones also tended to have a high enzyme activity.

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We found a nuclear RA-binding activity by using a sucrose-density-gradient assay from rat liver and testis. From the sedimentation analysis, and the comparison with cloned RARs, it is likely that these binding activities represent endogenous RARs. Furthermore we showed that these binding activities were constant irrespective of the retinoid status in the rat.

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