Biosynthesis of steroidal plant hormones, brassinosteroids, was studied using the cell culture system of Catharanthus roseus. Feeding labeled compounds of possible intermediates to the cultured cells, followed by analyzing the metabolites by gas chromatography-mass spectrometry disclosed the pathways from a plant sterol, campesterol, to brassinolide. There are two pathways, named the early C6-oxidation pathway and late C6-oxidation pathway, both of which would be operating in a wide variety of plants. Recent findings of brassinosteroid-deficient mutants of Arabidopsis and the garden pea by several groups, and the possible blocked steps of the mutants in the biosynthetic pathways are also introduced.

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To search for the most effective dietary n-3/n-6 ratio to suppress the type I allergic response, we performed basic experiments that applied parameters associated with the type I allergy. Guinea pigs fed on diets containing lipids with the n-3/n-6 ratio at different levels and the polyunsaturated fatty acid/saturated fatty acid ratio of a fixed level were sensitized with ovalbumin and reared for two weeks. The lowest or critical level of the n-3/n-6 ratio which produced a significant difference in the parameters was as follows: about 2.0 for the response of mast cells and eosinophils; 0.5 and 1.0, respectively, for the uptake of n-3 and n-6 polyunsaturated fatty acids and decreased histamine production; and 0.2 for decreased leukotriene B₄ and total leukotrienes 4, and increased leukotrienes 5/leukotrienes 4. The critical level of the n-3/n-6 ratio thus differed widely according to the parameter. Overall, the upper limit for the dietary n-3/n-6 ratio to suppress antigen-induced type I allergic responses is suggested to be around 1.0.

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We cloned and sequenced a 2.8-kb SalI fragment of Azospirillum lipoferum FS as a homologue of the Klebsiella oxytoca nifA gene. The amino acid sequence deduced from an open reading frame of 1872 bases showed 91% identity to that of the A. brasilense NifA, and the putative central σ⁵⁴ interaction domain was conserved as well as the C-terminal DNA-binding domain. The NifA function on the nifH promoter was examined in Escherichia coli using a combination of a nifA driver plasmid and a nifH-lacZ reporter plasmid, in which the transcriptional activation of the nifH promoter by the NifA was evaluated with the β-galactosidase activity. The A. lipoferum NifA activated the nifH promoter solely under microaerobic conditions, while the K oxytoca NifA activated it irrespective of the oxygen condition. These observations suggest that oxygen sensitivity is an intrinsic property of the A. lipoferum NifA.

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Collagen-related peptides, Gly-Pro-Arg and its analogues, were examined for their inhibitory effects on platelet aggregation induced by the addition of ADP. Human platelet aggregation was suppressed by more than 50% with each of Gly-Pro-Arg and such Gly-Pro-Arg-containing peptides as Gly-Pro-Arg-Gly, Gly-Pro-Arg-Gly-Pro, Gly-Pro-Arg-Pro-Pro, and Gly-Pro-Arg-Pro-Pro-Pro at a concentration of 0.3 mM. The inhibitory effects of these peptides were about 10 times higher in human PRP than in rat PRP. Other Gly-Pro-Arg analogues such as Sar-Pro-Arg, Gly-Pro-Lys, Gly-Ala-Arg, and Ala-Gly-Pro-Arg had no inhibitory effect at a concentration from 0.1 to 0.8 mM even in human PRP. Intravenous and oral administrations of Gly-Pro-Arg and enzymatic hydrolysates of collagen suppressed the decrease in platelet count for endotoxin-induced DIC in rats. Collagen itself has been regarded as a potent inducer of platelet aggregation, but these findings suggest that collagen-related peptides and enzymatic hydrolysates of collagen prevent platelet aggregation.

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Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis added to a culture of LLC-PK1 cells inhibited cell growth by 40%. In contrast with normal cells, the cells cultured in the presence ote PI-PLC showed needle-like appendages which seemed to have been formbd due to portions of the cell remaining adhered to the culture dish as the cell shrank. When LLC-PK1 cells were treated with PI-PI/C, significant amounts of alkaline phosphatase and alkaline phosphodiesterase I were released specifically from the apical surface of the LLC-PK1 cells. Furthermore, PI-PLC treatment caused a delay of enzyme production and dome formation. These data indicate that glycosyl-phosphatidylinositol (GPI)-anchored proteins on the surface of LLC-PK1 cells are important in cell growth and differentiation. Also, the combined use of LLC-PK1 cells and PI-PLC of B. thuringiensis is effective for investigating the function of GPI-anchor proteins.

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The BrCN cleavage of lactoferrin-a or -b (LF-a or LF-b) led to the observation of four fragments by SDS-PAGE, whose molecular masses were 77, 58, 52, and 30 kDas, or 74, 54, 47, and 30 kDas, respectively. N-Terminal amino acid sequence analyses show that the sequences of 58, 52, and 30 kDa fragments (residues 64-471, 130-471, and 472-689) of LF-a coincide with those of the 54, 47, and 30 kDa fragments of LF-b, respectively. All these fragments, which were positive by PAS staining, were not stained after being treated with glycopeptidase F. This treatment changed the 58 and 52 kDa fragments of LF-a to the 54 and 47 kDa fragments, respectively, whose molecular masses were the same as those of the treated fragments of LF-b. The 58 and 52 kDa fragments of LF-a bound to the lectin, Ricinus communis agglutinin, while the 54 and 47 kDa fragments of LF-b hardly bound to it.

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Changes in the heat-induced gelation properties of reconstituted rabbit skeletal actomyosin stored under a high salt concentration at pH 6.0 and 0°C were investigated at different weight ratios of actin to myosin by using dynamic rheological and biochemical measurements. The addition of actin resulted in a pronounced peak maximum at about 50°C and an accompanying temporary reduction in the range at about 50°C to 60°C. The more the initial actin concentration was increased, the greater was the area of the peak/shoulder. However, this area was markedly diminished with increasing storage time. As a result, the dynamic rheological pattern was transformed from an actomyosin type into a myosin type. The relationship between the G' value at 80°C and the actin/myosin weight ratio was curvilinear, with a peak at the ratio of 0.05, immediately after storage was started. This profile changed during storage, depending on the extent to denaturation of actin and myosin in the reconstituted actomyosin (RAM). The G' value of actomyosin in 0.5 M KCl with a small actin/myosin ratio of 0.05 decreased to one-half of its initial value after 7 days of storage, whereas the G' value with a large actin/myosin ratio of 0.225 increased by about 1.6 times. In 1.5 M KCl, all the G' values declined to the level with myosin alone after 7 days of storage. The time-course plots of the remaining actin concentration in RAM at different weight ratios of actin to myosin after being treated with 0.5 M or 1.5 M KCl showed a decrease in the actin content with increasing storage time, and an increase in the KCl concentration to 1.5 M KCl promoted the denaturation of actin in RAM faster than with 0.5 M KCl. The surface hydrophobicity of each RAM sample progressively increased with increasing storage time, while little significant increase in the sulfhydryl (SH) content during storage was observed. It is concluded that changes in the heat-induced gelation properties of actomyosin during storage are largely attributable to the denaturation of actin rather than to the denaturation of myosin or to quantitative changes in the SH content and hydrophobicity.

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The degradation process of β-conglycinin, a vicilin-type glycosylated storage protein of soybean seeds, during germination and seedling growth was examined by concanavalin A blotting combined with polyacrylamide gel electrophoresis. We detected and analyied the structures of key intermediary fragments of 30 kDa derived from β-conglycinin degradation, they were proved to be single-domain type subunits of β-conglycinin. We show here a degradation process of β-conglycinin: β-conglycinin is subjected to limited proteolysis at exposed regions on the molecular surface, like domain junctions, generating 30-kDa singledomain fragments before non-specific proteolysis.

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The gene, mdlA, coding for mono- and diacylglycerol lipase from Penicillium camembertii U-150 was expressed efficiently in Aspergillus oryzae under the control of its own promoter. The gene product was secreted into the culture medium with a highest productivity of 1g/liter and correctly processed at both N- and C-termini. KEX2-like processing was suggested to occur at the C-terminus in both A. oryzae and P. camembertii. Specific activity and substrate specificity of the purified recombinant protein were also almost the same to that of native protein but the extent of N-glycosylation in the recombinant protein was about half of that of the native protein. The presence of introns did not seem to affect the gene expression. The mdlA expression was induced by lipids and regulated transcriptionally in A. oryzae as well as P. camembertii. Promoter deletion analysis showed that the region between the positions at -382 and -554 bp from the translation initiation point was important to the higher expression of mdlA. The promoter sequence of mdlA was compared to that of the Geotrichum candidum lipase gene, which is also reported to be inducible by lipids, with three commonly observed oligonucleotide sequences.

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A series of 24a-substituted milbemycin A₄ derivatives were synthesized from 24a-hydroxymilbemycin A₄, which had been obtained by the microbial oxidation of milbemycin A₄. The acaricidal activity of each synthesized derivative against Tetranychus urticae was tested and some of the derivatives showed higher activity than parent milbemycin A₄. Among them, 24a-methylmilbemycin A₄ (22) was the most active derivative, with 100% mortality of the mite at a concentration of 1 ppm and 50% mortality at a concentration of 0.1 PPm.

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An affinity matrix of soluble starch gel was prepared by cross-linking catalyzed by epichlorohydrin. The elution pattern of Taka-amylase A (TAA) indicated that the amount of enzyme bound to the starch gel column increased with increases in the ammonium sulfate (AmS) concentration in the equilibrating buffer. TAA had an affinity for the gels with a starch structure, and desorbed from the column with the buffer containing no AmS. Bound TAA was also eluted with starch and cyclodextrin solution. The AmS stimulative effect was partially replaced by polyethylene glycol and surfactants. Besides TAA, various other amylases bound satisfactorily to the starch gel. Moreover, affinity purifications of dextranase, cellulase, and pectinase were done by gels with dextran, cellulose, and pectin structures, respectively. By the aid of forced effects of AmS, various carbohydrases could be purified by the affinity gels of polysaccharide linked by epichlorohydrin.

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We investigated the lipid peroxidation in linoleic acid micells caused by H₂O₂ in the presence of metmyoglobin by monitoring the oxygen consumption. O₂ consumption usually consisted of two phases. In the first phase, it occurred slowly and linearly until the concentration of linoleic acid hydroperoxide reached a certain value, rapid consumption, presumably by a chain reaction, then followed in the second phase. No effects of diethylenetriaminepentaacetic acid (DTPA) on the induction period (the period during the first phase) and the maximum oxygen consumption rate (MOCR) in the second phase indicate that free ferric ions liberated from myoglobin had no role in any phases during the lipid peroxidation. The differing dose effects of ascorbic acid, α-tocopherol, and sodium nitrite on the induction period and MOCR reflect their respective antioxidative mechanisms during lipid peroxidation.

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To investigate the transformation from (2E, 6E)-farnesyl pyrophosphate to (2Z, 4E)-γ-ionylideneethanol in the abscisic acid-producing fungi, Cercospora cruenta, plausible [2-¹⁴C]-C₁₅ intermediates were prepared and fed. Substrates such as (2E, 6E)-farnesyl pyrophosphate, (2z, 4E)-γ-ionylideneethanol and its pyrophosphate were incorporated into ABA and its known biosynthetic precursors. It is suggested that (2E, 6E)-farnesyl pyrophosphate is converted to (2Z, 4E)-γ-ionylideneethanol in four consecutive steps: dehydrogenation, isomerization, cyclization and hydrolysis.

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Mannosylated derivatives of cyclodextrins (CDs), mannosyl-α, β, and γCD were synthesized from a mixture of mannose and α, β, and γCD by the reverse action of α-mannosidase from jack bean, respectively. Their structures were analyzed by FAB-MS and ¹³C-NMR spectroscopies, and they were identified as 6-O-α-D-mannosyl-α, β, and γCD. The optimum conditions for the production of 6-O-α-D-mannosyl-αCD by α-mannosidase were examined. Optimum pH and temperature were pH 4.5 and 60°C, respectively. Yield of mannosyl-αCD increased with increasing mannose concentration and reached more than 35% (mol/mol) at the concentration of 2 M mannose and 0.4 M αCD.

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We developed a novel approach for the high-level production of a microbial transglutaminase (TGase) from Streptoverticillium in E. coli. The direct expression of the TGase gene in E. coli cells did not cause overproduction, probably due to the harmful influence of TGase activity, which introduces covalent crosslinks between proteins. Therefore, we fused the chemically synthesized TGase gene coding for the entire 331 amino acid residues at the amino terminus to a bacteriophage T7 gene 10 leader peptide (260 amino acids) using an inducible expression vector. The TGase gene was expressed as inclusion bodies in the E. coli cytoplasm. Restoring 15 amino acid residues upstream of the amino terminus of the mature TGase by a two-step deletion of the fusion sequence facilitated solubilization and subsequent proteolytic cleavage, thus releasing mature TGase. Although the mature form had less TGase activity than native TGase, because of the poor refolding rate, these results suggest that this system is suitable for the efficient production of TGase.

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The effects of sesaminol and sesamin on the ethanol-induced modulation of immune indices related to food allergy were examined in rats given a low (10%)-casein diet. Chronic ethanol drinking, at the dietary level of 23% (w/w), significantly increased the plasma IgA and IgM concentrations, irrespective of the presence of 0.1% and 0.2% sesaminol, but the effects disappeared with 0.2% sesamin. A significant IgG-elevating effect of these lignans was also found. In contrast, the concentration of plasma IgE was not influenced by the dietary manipulation. Although ethanol drinking did not influence splenic leukotriene B₄ production, sesaminol tended to decrease it dose dependently, while sesamin increased the plasma prostaglandin E₂ concentration. These results suggest that sesaminol and sesamin seems to have a diverse effect on the plasma levels of immunoglobulins and eicosanoids.

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To improve the efficiency of the enzymatic conversion of 5'-xanthylic acid (XMP) to 5'-guanylic acid (GMP), we attempted to increase the activity of the conversion enzyme, XMP aminase (GMP synthetase) encoded by the guaA gene in Escherichia coli. By connecting the P_L promoter of λ phage, the SD sequence of trpL of E. coli, and ATG, at a suitable position upstream of the guaA gene, we obtained plasmid pPLA66. Sequencing of the nucleotides of the upstream region of the guaA gene on pPLA66 showed that the C-terminal region of the guaB gene, which encodes IMP dehydrogenase, was conserved and a short peptide consisted of 14 amino acids was coded. E. coli MP347/pPLA66 showed an increase in the activity of approximately 370 times when compared with that of the strain MM294, and the amount of the enzyme protein represented approx. 34% of the total cellular protein. Strain MP347/pPLA66 was cultivated in a 5-liter jar fermentor using a medium which contained mainly corn steep liquor. The culture broth had high XMP aminase activity. In the conversion reaction using mixed broths consisted of 600ml of XMP-fermentation broth of Corynebacterium ammoniagenes KY13203 and 30ml of cultured broth of E. coli MP347/pPLA66, a surfactant, Nymeen S-215 and xylene were added to the reaction mixture to make the cell membrane permeable to nucleotides. After 23h of the reaction, 70mg/ml (131mM) of GMP·Na₂·7H₂O was accumulated from 83 mg/ml (155 mM) of XMP·Na₃.7H₂O, without addition of ATP. The molar conversion yield was approx. 85%. The facts that the cell membrane was treated to allow nucleotides to permeate and that the conversion reaction proceeded well enough in spite of a small amount of E. coli cells indicate ATP was regenerated from AMP by C. ammoniagenes cells and supplied to E. coli cells. Therefore, it was considered that the coupling reaction between these two kind of strains was established.

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Staphylococcal γ-hemolysin consists of two protein components, F (or HγI) and HγII. To elucidate the mode of action of γ-hemolysin, we studied the binding order of F and HγII to human erythrocytes and the cell-bound state of the two components. The binding of F to human erythrocytes preceded the binding of HγII to the cells, and thereafter hemolysis occurred. Western immunoblot analysis of the cell-bound γ-hemolysin indicated that F and HγII components form high-molecular-mass (150-250 kDa) complexes on the erythrocytes. The toxin complexes were recovered in a Triton X-100-insoluble fraction of the erythrocytes, which contains cytoskeleton proteins. Neither the formation of the toxin complex(es) nor hemolysis occurred when the erythrocytes were treated with proteinase K. Abortion of the complex formation on the proteinase K-treated erythrocytes may be due to the failure of the binding of HγII to the cells, because F bound to the proteinase K-treated erythrocytes to the same extent as to the non-treated erythrocytes.

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From microorganisms growing on a cell-wall preparation (CWP) of Schizophyllum commune as a carbon source, Bacillus circulans KA-304 was isolated on the bases of activities in culture filtrate to decrease turbidity of the CWP-suspension and to form protoplasts from S. commune. The culture filtrate was also active in hydrolyzing p-nitrophenyl (p-NP)-α-D-glucoside, p-NP-β-D-glucoside, and p-NP-β-D-N acetylglucosaminide. The protoplast-forming and the p-NP-glycoside-hydrolyzing activities were increased by the addition of CWP of S. commune to the culture medium, and this was not observed for other bacteria tested (15 genera, 80 species). B. circulans KA-304 was shown on gel filtration to produce at least two enzyme species for hydrolyzing both p-NP-β-D-glucoside and p-NP-α-D-glucoside. The protoplast-forming activity was retained for at least 6 months at 5°C as an ammonium sulfate (90% saturation) precipitate or at -20°C as a freeze-dried preparation (KA-preparation). The activity was stable at pH 6.5-7.0, and remained after 10 min of treatment at 40°C. Protoplast formation proceeded optimally at pH 6.5 with 50 mM potassium Phosphate buffer and 0.5 M mannitol as an osmotic stabilizer. B. circulans KA-304 seems to be a suitable strain producing enzyme(s) to prepare protoplasts from S. commune.

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A dipeptidyl carboxypeptidase (DCP) activity was detected in cell-free extracts of Pseudomonas sp. WO24. After purification and characterization the enzyme was found to be homogeneous by SDS-PAGE, and had a molecular mass of 74, 000 Da by SDS-PAGE and 72, 000 Da by gel filtration, indicating that it is monomeric. The isoelectric point was 5.2 and optimum pH was 6.5-7.0. It showed a specific activity of 780 μmol/min/mg, which is the highest of the values shown by known enzymes. The enzyme hydrolyzed angiotensin I to angiotensin II and sequentially released Phe-Arg and Ser-Pro from the C-terminus bradykinin. The DCP could not cleave imido-bonds, Gly-Gly bonds, or tripeptides. The enzymatic activity was completely inhibited by 0.001 mM EDTA and 0.1 mM o-phenanthroline, but it was not affected by general serine and cysteine protease inhibitors. Addition of Zn²⁺ completely restored the original activity of the inactivated DCP treated with EDTA. These results suggest that this enzyme is a zinc metalloprotease. The characteristics of the purified enzyme are slightly different from those of the DCPs from Escherichia coli, Pseudomonas maltophilia, and Corynebacterium equi, and considerably from those of the DCP from Bacillus pumilus.

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Three-day-old rice seedlings treated with ethylene showed elongation of the 2nd and 3rd leaves. This ethylene-stimulated elongation was not observed in the presence of uniconazole-P or prohexadione, both gibberellin (GA) biosynthesis inhibitors, suggesting that GA was involved in the response. An analysis of endogenous GAs by GC-MS revealed that the GA₁ level was reduced in the 3rd leaf in response to ethylene. Dose-response experiments showed that the responsiveness to GA₁ was enhanced by ethylene. Feeding experiments of ¹⁴C-GA₁ with ethylene-treated seedlings showed that ethylene may increase the conversion of GA₁ to GA₈. These results suggest that, in young seedlings of rice, ethylene stimulates leaf elongation by increasing the responsiveness to GA₁ and the turnover of GA₁.

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We searched our chemical collection to identify non-N-acetylneuraminate (NeuAC) inhibitors of influenza neuraminidase (NA). Of the 62 acyl derivatives tested, several acyl amino acids, but not acyl alkanolamine derivatives, were effective and inhibited the NA activity in a dose-dependent manner. N-3-Hydroxymyristoyl D-cysteine and N-myristoyl-O-caproyl-D-serine were the more potent compounds and inhibited the enzyme in a noncompetitive manner (K_i=102 and 125 μM, respectively) without respect to the substrate. An important consideration for the choice of inhibitor is the selectivity of the inhibition. These compounds were selective inhibitors of viral NA and effective for any variant enzyme, but the enzymes from V. cholerae and human placenta were insensitive. Accordingly, the acyl amino acid derivatives may be expected to be inhibitors without cellular toxicity and may serve as lead compounds for anti-influenza agents.

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A cDNA encoding sugar beet α-glucosidase was cloned from a library constructed from mRNA of suspension-cultured cells. The cDNA, 3056bp in length, had an open reading frame encoding a polypeptide of 913 amino acid residues with a molecular mass of 102, 078 Da, included only one of four regions which were conserved in the α-amylase family of enzymes. The deduced amino acid sequence from the analysis of the cDNA contained the sequences of the proteolysis peptides and the active site region peptide of sugar beet α-glucosidase. The primary structure indicated relatively high homology in the range of 28.2 to 54.3% to those for other α-glucosidases. The highest homology was found in barley α-glucosidase.

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The absolute configurations of some hydroxy-fatty acids were examined by the modified Mosher's method proposed by Ohtani et al. The absolute configurations of the major components were determined from NMR data of their MTPA esters and 2-NMA esters. The application of Mosher's method for the anti-glycol is discussed.

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Bacteriocins produced by Lactobacillus acidophilus JCM 1023, JCM 1028, JCM 1021, JCM 1229, and JCM 5342 were active against closely related lattobacilli. These bacteriocins were purified and partial sequenced. Bacteriocin activities of L. acidophilus JCM 1023 and JCM 1028 were associated with two components. On the basis of N-terminal amino acid sequencing and tbe molecular masses, it is interpreted that these two-component bacteriocins are identical to acidocin J1132, a bacteriocin from L. acidophilus JCM 1132 [Tahara et al., Appl. Environ. Microbiol., 62, 892-897 (1996)]. Other bacteriocins were single-peptide bacteriocins.

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The effect of pancreaticobiliary secretion on the intestinal expression of the apo A-IV gene was examined in fasted rats. Pancreaticobiliary diversion, but not biliary diversion alone, into the ileum increased the ileal apo A-IV mRNA expression by 24 h post-operation. Jejunal apo A-IV mRNA was reduced by biliary exclusion. The data suggest that the biliary constituent plays an important role in the apo A-IV gene expression in the entire length of the small intestine, and that up-regulation of the apo A-IV gene requires exocrine pancreatic in addition to biliary secretion.

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Purpurogallin showed antibacterial activity toward gram-positive bacteria. Strong activity against methicillin-resistant Staphylococcus aureus [minimal inhibitory concentration (MIC) against methicillin of 1600 μg/ml] was found, with MIC of 11.0 μg/ml. Purpurogallin inhibited the growth of all tested plants and decreased the chlorophyll content in the cotyledons of Brassica campestris subsp. rapa. It showed potent inhibitory activity against prolyl endopeptidase (the 50% inhibitory concentration was 1.6×10^-5 M), unlike its analogues, hinokitiol and tropolone.

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A glutaraldehyde (GA) spacer complex on enzyme immobilization was cleaved from the support introduced N^α-9-fluorenylmethyl-oxycarbonylglycine. By the ¹H-NMR spectroscopic measurement of the desired complex, glycin-GA-phenethylamine (GGP), GA was thought to be involved in enzyme immobilization as a heteropolymer with the molecular weight range of about 600 to 1300.

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To analyze cobalamin metabolism in photosynthetic green algae, the effects of cobalamin on growth of Chlorella vulgaris C-30 were studied and the algal cobalamin contents were assayed. Cobalamin significantly stimulated growth of the Chlorella cells, but biologically inactive cobalamin analogues did not. Chlorella grown in a cobalamin-free medium (control) contained cobalamin coenzymes, 5'-deoxyadenosylcobalamin (7.95±0.31 ng/g wet weight) and methylcobalamin (2.72±0.45 ng/g wet weight), of which the levels were increased significantly in cobalamin-supplemented cells. These results indicate that the alga has ability to take up exogenous cobalamin and synthesize the coenzyme forms.

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A thermostable trehalose synthase from Thermus aquaticus ATCC 33923, which catalyzes the interconversion between maltose and trehalose by intramolecular transglucosylation, converted sucrose into trehalulose (1-O-α-D-glucopyranosyl-D-fructose). The trehalulose-forming activity of the enzyme was very low compared with that of maltose and trehalose. Kinetic studies showed that sucrose competitively inhibited the interconversion activity between maltose and trehalose. Consequently, these three substrates, maltose, trehalose, and sucrose, are thought to bind the same active site of trehalose synthase.

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The selective incorporation of linoleic (18:2(n-6)) and docosa-hexaenoic (22:6(n-3)) acids into phospholipids of mitochondria, endoplasmic reticulum, and plasma membrane was investigated by changing the ratio of 22:6(n-3) against 18:2(n-6) in a medium, in which Chinese hamster V79-R cells were grown. In those organelles, 18:2(n-6) and its elongation product (eicosadienoic acid) (20:2(n-6)) were predominantly incorporated into phosphatidylcholine. However, 22:6(n-3) was incorporated more selectively into phosphatidylethanolamine than 18:2(n-6) and 20:2(n-6).

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Mutastein, a casein polymer that inhibits insoluble glucan synthesis by Streptococcus sobrinus, was produced when Aspergillus terreus was grown in a medium containing α-casein. In these experiments, it was shown that flavipin (3, 4, 5-trihydroxy-6-methyl-o-phthalalde-hyde) isolated from cultures of A. terreus caused polymerization of α-casein. Like mutastein, the polymerization products were active in inhibiting insoluble glucan synthesis by S. sobrinus.

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NP220s compose a family of RNA binding proteins together with matrin 3, one of major proteins of the nuclear matrix. They have repeats of RNA recognition motif (RRM; MH2) homologous to RRM in heterogeneous nuclear RNPs I/L in addition to MH1 and MH3 with unknown function. In search of additional homologous sequences, we found the reported sequence of rat matrin 3 is partially incorrect. Correction of this sequence showed that the NP220 family has a fourth homologous motif with the characteristics of a Cys₂-His₂ zinc finger-like motif. The sequence of this motif is perfectly conserved in human and mouse NP220s despite their 75% overall sequence homology.

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The level of mRNA for endo-1, 4-β-glucanase was increased before the exponential phase of growth and decreased rapidly during the exponential phase in suspension-cultured poplar cells. The level of mRNA was increased for a short period after the addition of either 2, 4-D or sucrose to the culture medium at the stationary phase. The level was increased to the maximal level when both 2, 4-D and sucrose were provided together, but one did not increase the effect of the other. These findings suggest that expression of gene encoding poplar endo-1, 4-β-glucanase is controlled during cell growth by independent systems activated by auxin and sucrose.

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A gene encoding a homolog of the Streptomyces chymotrypsin-like serine protease, SAM-P20, was identified downstream of the sam-p20 gene and designated SAM-P20D. This gene has two tandem Shine-Dalgarno sequences and two initiation codons. We have established vector systems with the function of tyrosinase gene-bone melanin pigmentation as a reporter for sam-p20D gene expression in Streptomyces coelicolor in order to identify the promoter and terminator activities. Using this system, the sam-p20D gene was suggested to be transcribed monocistronically.

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Penicillium sp. NTC-47, which had been isolated from a soil sample, produced novel dihydroquinolinones when cultured with okara (the insoluble residue of whole soybean). These metabolites, 1 and 2, were crystalline products with molecular formulas of C₁₇H₁₇NO₅ and C₁₇H17_</SUB>NO₄, respectively. The structure of 1 was established to be 3-methoxy-4, 5-dihydroxy-4-(4'-methoxyphenyl)-quinolinone by spectroscopic evidence and by an X-ray crystallographic analysis. Spectral data indicated the structure of 2 to be 5-deoxy-1. Compound 1 demonstrated toxicity against brine shrimp with an LC₅₀ value of 20 μg/ml, but 2 exhibited no activity at a dose of 100 μg/ml.

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Archaeal 16S rDNA clones retrieved from paddy soil DNA were sequenced. Among 100 clones, 88 clones were assigned to methanogens and nine clones were assigned to crenarchaeota. However, three of the nine clones were phylogenetically far from the cultured crenarchaeota and closely related to marine planktonic archaea. The other three clones showed extremely novel 16S rDNA sequences and were phylogenetically far from both Crenarchaeota and Euryarchaeota. This paper reports the ubiquitous presence of crenarchaeotal and extremely novel clones in paddy soils.

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Grandinal, a new dimeric phloroglucinol compound, was isolated from Eucalyptus grandis and characterized by spectral techniques. Tautomeric structures 1, 2, and 3 were assigned to grandinal. Biogenetically, 1 is proposed to be formed from intermediates derived from grandinol and jensenone.

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Free N-glycans have been found in pea seedlings. These free N-glycans were coupled with 2-aminopyridine and purified by gel filtration, Con A-Sepharose affinity chromatography, and size fractionation HPLC. These structures of pyridylaminated free N-glycans were analyzed by exomannosidase digestions and ionspray tandem mass spectrometry. The structural analyses showed that the several oligomannose-type sugar chains having one GlcNAc residue at the reducing-end side occur in the seedlings, suggesting the endo-β-N-acetylglucosaminidase PS [Y. Kimura et al., Biosci. Biotech. Biochem., 60, 228-232 (1996)] should be involved in the release of oligomannose-type N-glycans from the storage glycoproteins [Y. Kimura et al., Biosci. Biotech. Biochem., 60, 1841-1850(1996)] during the germination of pea seeds.

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A Caenorhabditis elegans cDNA for a glutamate transporter was cloned and examined in this study. The predicted protein is 11 residues shorter at the N-terminus than Ceglut-1, which we previously reported. The protein, when expressed in Xenopus laevis oocytes, showed much higher glutamate transport activity than Ceglut-1, suggesting that the N-terminal sequence is critical in glutamate transport.

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