Bacterial plasmids of Escherichia coli incompatibility group Q (IncQ) are broad-host-range plasmids that are able to proliferate in almost all Gram-negative bacteria. They are small, nonconjugative, and multicopy plasmids. They can be mobilized into many species of Gram-negative bacteria by coresident conjugative plasmids. Plasmids RSF1010, R1162, and R300B have DNAs of a size of 8.7 kb, and are best studied among IncQ plasmids. These plasmids encode by themselves three major proteins essential for the initiation of DNA replication. This makes the plasmid DNA replication less dependent on the DNA replication apparatus of host cells, and leads to promiscuity or a broad host range. Considering the biological features of these plasmids, they are potent DNA cloning vehicles. Moreover, their characteristic DNA replication mechanism that makes IncQ plasmids promiscuous is elaborate, and is an interesting object of scientnfic studies.

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We used a plasmid carrying a sequence for autonomous maintenance in Aspergillus (AMAl) and the E. coli uidA gene as a reporter gene to search the A. oryzae and A. niger genomes for DNA fragments having strong promoter activity. β-glucuronidase (GUS)-producing A. oryzae transformants containing the No. 8AN derived from A. nigei, or the No. 9AO derived from A. oryzae, were constitutive for the expression of the uidA gene when cultivated in the prcdence of a variety of carbon and nitrogen sources. When the GUS-producing transformants were grown in liquid culture, the No. 8AN showed an increase of approximately 3-fold in GUS activity compared to the amyB (α-amylase encoding gene) promoter. There was also a correcponding increase in the amount of GUS gene-specific mRNA. When these transformants were grown as rice-koji, the No. 8AN showed an increase of approximately 6-fold compared to the amyB promoter, and the amount of GUS protein produced also increased. These strong promoter regions might be applicable to the production of other heterologous proteins in Aspergillus species.

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The zjmograms of peroxidase (POD) and esterase (EST) from seventy-two kinds of Citrus fruits and the fruit of both Poncirus and Fortunella were obtained by polyacrylamide gel electrophoresis. It was found that there was little variance in the band pattern of POD and EST of the fruits harvested after September. There were at least three bands of POD and approximately ten of EST in each cultivar. The density of each band for EST was measured with a densitometer. There was a characteristic band for POD identifying pummelos. The band patterns of EST were also characteristic among each species or cultivar. Two kinds of ootachibana (Citrus otachibana Hort. ex Tanaka) were shown to be different cultivars from each other according to their EST zymograms and peel oil analysis. Cluster analysis based on EST was done and discussed together with POD.

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Two forms of the restriction enzyme HindIII were alternated with each other under some physiological or biochemical conditions. Addition of a low amount of phase T7 to the culture of HindIII-producing Haemophilus influenzae Rd, resulted in appearance of some amounts of the P2 fraction of HindIII, which was eluted with a high concentration of KCl from a phosphocellulose column. Higher amounts of T7 caused a decrease of the P2 fraction; finally the alternative P1 fractioni of HindIII, which was eluted with a lower concentration of KC1, remained exclusively. Addition of disaccharides such as maltose and trehalose to the bacterial extract, yielded more P2, although the disaccharides inhibited to this enzyme. Urea showed an interesting distribution of these two forms of HindIII. Phosphocellulose chromatography in the presence of 2M urea generated a broad peak of HindIII Activity. Addition of 4M urea, on the contrary, showed only one active peak of this enzyme. The HindIII could be purified by the following DEAE-cellulose chromatography. These results indicate the presence of only one kind of HindIII molecule, which was alternated between free and bound forms, and a certain kind of factor that would equilibrate these two forms.

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Cutinostatin B was purified from a culture filtrate of a strain No.3112-4 of an actinomycete which had been isolated from a soil sample collected in Setagaya-ku, Tokyo, Japan. The structure of this compound was determined mainly by 2D NMR experiments to be N^α-[N^α-(2, 3-dihydroxybenzoyl)-ornithyl]-N^δ-hydroxyornithine-δ-lactum. Cutinostatin B inhibited the cutinase activity of Cladosporium fulvum when using β-naphthyl caproate as the substrate with an IC₅₀ value of 28.9μM (11.0μg/ml), and inhibited 95% when using crude apple cutin as the substrate with a concentration of 263μM (100μg/ml).

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Structures are proposed for the Inagami-Tamura endogenous digitalis-like factor (EDLF), and two possible candidates, (14β, 15β, 16β, 17α)- and (14β, 15α, 16α, 17α)-2, 3, 14, 15, 16, 17-hexahydroxy-1, 3, 5(10)-estratrienes, were synthesized. Both compounds were potent in inducing a contractile response in isolated rat aorta and guinea pig left atrium.

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Structure 1 is proposed for the Inagami-Tamura endogenous digitalis-like factor (EDLF), and (14β, 17α)-14-hydroxy- and (14β, 17α)-2, 14-dihydroxyestradiols (2 and 3) were synthesized as model# for studies on 1. The latter compound was remarkably potent in inducing a contractile response in isolated rat aorta and guinea pig left atrium.

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To produce catalase-free uricase preparations, we constructed catalase-deficient strains from Escherichai coli MC1000 and MM294 and used them as recombinant host strain. The parent strains and catalase-deficient strains showed no differences in the growth characteristics by shaking culture in Erlenmeyer flasks. The catalase deficient strain derived from MC1000 transformed with the uricase expression plasmid pUT118 (strain SN0037) had growth characteristics and the uricase productivity comparable to those of the parent host strain MC1000 in fed-batch culture in a jar fermentor and no catalase activity was detected in cell-free extracts. However, the katG disrupted strains from MM294 carrying pUT118 had poor growth and their uricase productivities were low compared to those of the parent strain MM294. Using the strain SN0037, a catalase-free uricase preparation was obtained with fewer purification procedures and the final recovery of uricase activity was improved. The catalase-deficient E. coli host strain will be a suitable host for the production of the uricase, free of catalase activity, in high yield.

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The recovery of soybean oligosaccharides from the steamed soybean waste water in tofu (soybean protein curd) processing was carried out by using reverse osmosis and nanofiltration membranes. The feed solution was prepared by isoelectric and ultrafiltration treatments. Concentrations of the total oligosaccharides of 10% (w/v) and 22% (w/v) were obtained by using the RO and Nf membranes in a batch operation. The chemical oxygen demand of the feed solution was simultaneously reduced from 8400-8700 ppm to 27-160 ppm. The permeate flux was mathematically analyzed by the osmotic pressure model with concentration polarization, the simulated results agreeing well with the experimental ones.

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We investigated the immunological function of cheede whey protein concentrate (CWPC), which is a by-product of cheese production, using mitogenic activity in murine splenocytes as an index. A fraction isolated by gel filtration and anion exchange chromatography of CWPC showed high mitogenic activity, comparable to the activity of lipopolysaccharide (LPS). The fraction was detected as a single band on SDS-PAGE. It contained calcium, inorganic phosphorus, and carbohydrate, indicating the active component to be a glycophosphopeptide (GPP). Since Pronase digestion of GPP did not reduce its mitogenic activity, carbohydrate rather than peptide may be important in the activity. When applied on an anti-β-caseinophosphopeptide (β-CPP) antibody aftinity column, the GPP was separated into two components, one with affinity to β-CPP and the other without such affinity. Both the components contained N-linked oligosaccharide chains and had the mitogenic activity. These results demonstrate that cheese whey contains a GPP having strong mitogenic activity.

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To study the effects of food additives on glutamate receptors, they were expressed in Xenopus oocytes that received an injection of poly(A)⁺mRNAs prepared from rat brain. The response of the receptors elicited by kainate (KA) and N-methyl-D-aspartate (NMDA) was measured electrophysiologically in the presence and absence of food additives. Both responses elicited by KA and NMDA were inhibited similarly by addition of additives such as caffeine, vanillin or saccharin. However, inhibition of KA-elicited response by food additives followed a competitive inhibition scheme with two binding sites, while that of NMDA-elicited response followed a simple noncompetitive inhibition scheme. Inhibition constants of food additives for both responses were more than 1mM. So it is unlikely that food additives taken with processed food interrupt signal transmission under physiological conditions. The specificity of inhibition of both responses was examined by adding various compounds to the bathing solutions containing the agonist. Increase of the number of hydroxyl groups in alcohols with the chain of three carbon atoms decreased the potency of inhibition. Potency of the inhibition depended on the species of functional groups. The order of potency of the inhibition by compounds with a chain of six carbon atoms was alcohol=diamine > aldehyde > carboxylic acid. Hexanol inihibited the receptors more strongly than (3Z)-hexen-1-ol. NMDA-elicited response showed little selectivity in inhibitioni by structural isomers of pentanol, while KA-elicited response showed some selectivity in inhibition by the structural isomers.

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This study was done to clarify the effects of dietary wheat gluten on the hepatotoxic action of D-galactosamine (GalN) and endotoxin (Etx). Male Wistar rats fed a high casein or high gluten (supplemented with L-Lys and L-Thr) diet were injected with GalN or Etx, and the plasma glutamate oxaloacetate transaminiase, glutamate pyruvate transaminase, and lactase dehydrogenase activities were examined 20h later. In rats fed the high gluten diet, these enzyme activities were lower than in the high casein group after injection of 800mg/kg of GalN. But such a difference between the casein and gluten groups was not clear when they were treated with 400 mg/kg of GalN nor observed even after injection of Etx or Etx + GalN (400mg/kg). Similarly these was no difference in the plasma concentrations of Etx, tumor necrosis factor-alpha, or interferon-gamma in the rats receiving an injection of 800mg/kg of GalN between both dietary groups. These results suggest that dietary gluten affords protection against hepatic injury by a high dose of GalN but not by a low dose of GalN and/or Etx.

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The AgeI restriction-modification system from a marine bacterium, Agrobacterium gelatinovorum IAM12617, recognizes the nucleotide sequence ACCGGT. The gene coding for the AgeI methylase (M.AgeI) was cloned into Escherichia coli DH5 αMCR, and the nucleotides of the gene were sequenced. The M.AgeI gene coded for a protein of 429 amino acid residues (molecular mass, 47, 358 daltons). The deduced amino acid sequence of M.AgeI was compared with those of other methylases and showed that there are high degrees of similarity in some cytosine-5 methylases.

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The Rhizopus niveus leul gene was cloned using a 2-kb HindIII fragment of the leuA gene of Mucor circinelloides as a hybridization probe. The coding region of this gene comprises 1890bp that encode a putative protein of 630 amino acids. The predicted amino acid sequence is similar to those of other α-isopropylmalate isomerases (α-IPMIs), showing 79.4%, 71.7%, and 60.6% identity with the Mucor circinelloides LeuA, Phycomyces blaskesleeanus Leu1, and Ustilago maydis Leu1, respectively. The vector pRN1, which contains a 3.7-kb BamHI-SacI fragment of the R. niveus leul gene, could complement leucine auxotrophy of R. niveus M-37 (leu1^-), suggesting that the gene codes for α-IPMI of R. niveus. Both the transformation frequency and mitotic stability with pRN1 were higher than those with leuA gene-containing vectors. It was found by using the leul Aene that the autonomously replicating sequence (ARS) of P. blakesleeanus partially functions as an ARS in R. niveus.

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A thiamine-binding protein was purified from rice germ (Oryza sativa L.) by extraction, salting-out with ammonium sulfate, and column chromatography. From the results of molecular mass, K_d and B_max values for thiamine-bindinig, binding specificity for thiamine phosphates and analog, the protein was suggested to be identical to the thiamine-binding protein in rice bran. The thiamine-binding protein was more efficiently purified from rice germ than from rice bran. The protein was rich in glutamic acid (and/or glutamine) and glycine. The protein did not show immunological similarity to thiamine-bindinig proteins in buckwheat and sesame seeds. However proteins similar to the thiamine-binding protein from rice germ existed in gramineous seeds. They were suggested to have thiamine-binding activity and to be of the same molecular mass as the thiamine-binding protein.

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The inhibition by melanoidin of trypsin was investigated in a kinetic approach. Melanoidin was prepared by the Maillard reaction between D-glucose and glycine, and BANA was used as a substrate. The inhibition was found to follow a non-competitive mode with a K_i of 5.8% and to be exerted through an electrostatic interaction between melanoidin and trypsin. Approximately 20% of the activity of trypsin survived even at a greatly increased concentration of melanoidin. The inhibiting mechanism of melanoidin might be due to an allosteric effect, which was probably different from a proteinaceous inhibitor such as ovoinhibitor diminishing trypsin-activity completely. The melanoidin molecule was thought to trap a great number of trypsin molecules, binding them and reducing their activity.

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We found a novel metalloproteinase, which has high activity at low temperatures and in the presence of organic solvents, in the culture supernatant of a marine bacterium, Vibrio sp. T1800. The metalloproteinase, named vimelysin, was purified from the dulture supernatant by three column chromatographies. About 150mg of purified vimelysin was obtained from 3.3 liters of the culture supernatant with a high yield of 57%. The purified vimelysin showed a single protein band on SDS-PAGE with molecular weight of 38, 000. The isoelectric point of vimelysin was 4.3 by isoelectric f6cusing. The optimum pH of vimelysin was pH 8.0 or pH 6.5 using casein or furylacryloyl-glycyl-leucine amide (FAGLA) as substrates, respectively. The optimum temperature of vimelysin was 50°C when cdsein was uSed as a substrate, but it was 15°C when FAGLA was used as a substrate. Interestingly, vimelysin activity was completely retained after 48h of incubation at 25°C in the presence of 50% ethanol. Moreover, vimelysin showed 40% activity of the control even in the presence of 10% ethanol, while thermolysin showed only 5% activity under the same conditions.

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The fate of Mn²⁺ inside Saccharomyces cerevisiae cells was monitored during import and export processes, using information from electron paramagnetic resonance (EPR) spectroscopy analysis. EPR spectra showed that when entering the cell in high number, manganese ions immediately precipitated. If recorded under conditions that favored manganese efflux, EPR spectra indicated that only osmotically free ions were exported and that bound manganese had to turn into the soluble form before being able to leave the cell.

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A plasmid pLC-bar containing the bialaphos resistance gene derived from Streptomyces hygroscopicus between the Lentinus edodes ras gene promoter and priA gene terminator was constructed. When protoplasts of Pleurotus ostreatus were mixed with the plasmid DNA in the presence of polyethylene glycol and CaCl₂, bialaphos-resistant colonies were obtained. This indicated that transformation was successful. Southern blot analysis of total DNAs from transformants showed that the introduced plasmid DNA was integrated into the host chromosome and partly rearranged. A plasmid, pLC-GUS, containing the Escherichia coli β-glucuronidase (GUS) gene under the control of the L. edodes ras gene promoter and priA gene terminator was constructed and introduced into protoplasts of P. ostreatus with pLC-bar by co-transformation. Two of 5 transformants obtained as bialaphos-resistant colonies showed two to twenty times higher specific activity of GUS than the recipient. Southern blot analysis of total DNAs from transformants indicated the presence of the GUS gene only in the two transfotmants. These results indicated that co-transformation of P. ostreatus was successful, and that the GUS gene was expressed in P. ostreaths. This trainsformation system will enable us to breed commercial strains of P. ostreatus at the molecular level.

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Several saccharides were found to be significantly effective in providing protection against hydrostatic pressure and high temperature damage in the yeast Saccharomyces cerevisiae. The extent of barotolerance and thermotolerance with seven different sugars showed a linear relationship to their mean number of equatorial OH groups. The same linear relatioship is seen when sugars protect protein molecules against elevated temperatures in vitio. Some sugars were more effective in providing protection against hydrostatic pressure nearly a hundred times than high temperature. Pre-heat shock treatment on yeast cells induce various stress tolerdnces. In this report, pre-heat shocked cells showed potent protection against elevated temperature, but these cells showed faint protection agdinst elevated presSure. These results suggest that sugars may protect cells against hydrostatic pressure and high temperature in a similar manner, probably by stabilizing the macromolecule(s), and such type of protection may be suited for pressure stress.

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A correlation between tyrosinase activity and gill browning during preservation of Lentinus edodes fruit-bodies was observed. Latent-type tyrosinase was recognized in the precipitate of the gill homogenate, and activation of the tyrosinase was brought about by incubating the precipitate with acidic buffer. Changes in latent- and active-type tyrosinase content during gill browning indicated the possibility of a de novo synthesis of latent-type tyrosinase.

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CB-1, a new antifungal, was piirified from Bacillus lichenifrormis. The molecular mass of CB-1 was estimated as 42 kDa by gel filtration column Chromatography. CB-1 seemed to be an aggregation Product from 4 kinds of peptides. CB-1 conitainied 10 amino acids and some fatty acids of C_14-18:0 and C_18:1. CB-1 irreversively bound with chitin powder.

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Alliin has been reported to have antioxidative activity. We tried to reexamine the antioxidative activity of alliin with a purified alliini preparation, and found that it has no anitioxidative activity in a linoleic acid oxidation system. The reducing activity of alliin tested by the ECD method was also weak in showing antioxidative activity.

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The products of reduction of 2-methylcyclohexanone by Aspergillus repens MA0197 were analyzed quantitatively by GC after conversion to corresponding diastereoisomeric (-)-menthyl carbonate derivatives. Although the contents varied considerably with time, the predominant production of S-alcohol was observed, that is, Prelog's rule was obeyed.

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Blood pressure increases were inhibited by feeding a diet containing sour milk fermenited by a starter containing Lactobacillus helveticus and Saccharomyces cerevisiae to spontaneously hypertensive rats. In rats fed with the sour milk, the angiotensin I-converting enzyme activity of the aorta was significantly lower than that of rats fed with the conitfol commercial diet.

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Cell-free extracts of the unicellular algae Chlorella pyrenoidosa induce a UV-dependent incision of both φ×174 RFI DNA and a 27-mer double-stranded oligonucleotide containing a single thymine dimer, as analyzed by agarose and denaturing polyacrylamide gel electrophoresis, respectively. The incision reaction occurred only on the thymine dimer-containing strand of the 27-mer, and a major incision product of 17-mer was observed. Addition of ATP or ATP-regenerating system to algal extracts increrised the effect of DNA incision.

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The induction of the chicken ovalbumin (OVA) gene by different classes of steroid hormones and the mRNA levels of estrogen (ER), progesterone (PR), and glucocorticoid (GR) receptors were studied in chick oviducts. Combined treatment with two hormones increased the induction of the OVA gene more than single treatment, when the levels of OVA mRNA were measured with Slot blot analysis. To discover the role of nuclear steroid hormone receptors as transcriptional factors in the OVA gene inductioni, we analyzed thelevels of ER (with RT-PCR), PR, and GR mRNAs (with Northern blottinig). The level of PR mRNA was increased only by estrogen, while no steroid hormone affected the levels of ER and GR mRNAS. Thus, these findings show that the levels of nuclear receptors do not reflect the OVA mRNA level in the oviduct of steroid hormone-treated chicks.

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A mutant carboxypeptidase Z from Absidia zychae in which Gly137 was replaced by Ala by site-directed mutagenesis was constructed and expressed in Saccharomyces cerevisiae YPH250. The mutant enzyme hydrolyzed C-tetminal Pro-X bonds (X=amino acid) more efficiently than the wild-type enzyme and sequentially released amino acids from the C-termini of oligopeptides.

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Streptomyces griseus produced a certain kind of flocculating substance(s). The flocculating substance(s) could aggregate suspended kaolin particles in solution to form small flocs and enables it to sediment those particles. The production of flocculating substance(s) by S. griseus was not parallel to the cell growth and a large number of flocculating substance(s) was released into the culture broth in the death phase. The flocculating substance(s) produced by S. griseus acted under acidic conditions and the maximum activity was observed at pH 4.

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Urinary excretion of N^τ-methylhistidine (MeHis) in rats was linearly elevated by starvation for 2days. Plasma concentration of MeHis on day 1 and day 2 of starvation were increased 2.4- and 2.6-fold, respectively. The amount of released MeHis from the isolated muscles into medium during a 2-h incubation period was increased with starvation corresponded to the plasma MeHis concentration. The results of this study suggest that plasma MeHis is a sensitive index of myofibrillar protein degradation.

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Cultured roots of Menispermum dauricum, a rich source of the bisbenzylisoquinoline alkaloid dauricine (1), were fed with L-[U-¹⁴C]tyrosine, L-[3-¹³C] tyrosine, and [2-¹³C] tyramine independently, and the incorporation of possible early precursors into 1 was studied. The results demonstrdted that 1 was composed of four molecules of tyrosine, and that tyramine was specifically incorporated into the isoquinoline portions of 1. The unusual chlorine-containinig alkaloid acutumine (3), into which ¹⁴C-labeled tyrosine was also incorporated, was identified as onie of the main conistituents in the alkaloid fraction from the roots.

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Hasakol, a new antispasmodic coumarin isolated from Citrus hassaku, was synthesized from 7-geranyloxycoumarin by an improved method. This Synthesis involves only 3 steps and results in a yield 27% based on the starting material.

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Two epimers of methyl jasmonate were optically resolved by capillary gas chromatography, using heptakis (2, 3, 6-tri-O-methyl)-β-cyclodextrin as the chiral stationary phase. In the tea volatile concentrates, both of these epimers were present as only one enantiomer, their absolute configurations being ascertained as (-)-(1R, 2R)-methyl jasmonate and (+)-(1R, 2S)-methyl epijasmonate. The thermal isomerization of methyl epijamonate to methyl jasmonate was also clarified by optically resolved gas chromatography to have occurred at the asymmetric carbon of the C-2 position that is connected to the carbonyl group.

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When Mucor javanicus was cultured with castanospermine, two α-glucosidases (CS-I, II) were produced. The two enzymes were purified by two CM-cellulofine column chromatographies, and compared with the α-glucosidase (C) from a culture without castanospermine. The molecular weights of the three enzymes were calculated to be 97, 000 (CS-I, II) and 94, 000 (C) from SDS-PAGE. CS-II was similar in hydrolysis of α-1, 4-linkage to C, but hydrolyzed α-1, 2-, α-1, 3-, and α-1, 6-linkages at a lower rate than C. The Michaelis constant of CS-I for malto-oligosaccharides was several times as high as the constants of CS-II and C, but CS-I hydrolyzed nigerose at a faster rate than maltose.

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The effects of astilbin in Kohki tea, which is produced from the leaves of Engelhardtia chrysolepis HANCE (Chinese name, huang-qui), and of an aglyconie of astilbin, taxifolin, on the serum and liver lipid concentrations, and on the erythrocyte and liver antioxidative enzyme activities were determined with rats fed on a cholesterol-free diet. The total liver cholesterol concentration tended to be decreased by feeding with astilbin, and significantly decreased by feeding with taxifolin. The liver phospholipid concentration was decreased by feeding with both astilbin and taxifolin. In addition, astilbin and taxifolin lowered the serum and liver TBARS concentrations, but did not influence the serum and liver antiokidative enzyme activities, suggesting the possibility that these compounds acted to lower the TBARS concenitration by their direct antioxidative action in vivo, almost without influencing the antioxidative enzyme activities.

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Synopsis Model systems composed of 2-hydroxycyclohexanone and ammonium acetate in a mole ratio of 1 to 3 were set up to study the formation of octahydrophenazine (OHP). Octahydrophenazine and an intermediate were isolated and characterized by GC/MS (EI and CI). This intermediate was the condensation pfoduct between a molecule of 2-hydroxycyclohexanone and 2-amino-cyclohexanone via the Schiff base formation mechanism. A larger amount of the intermediate than of OHP was found in the solvent system containing less than 20% ethanol, the relative proportion of the intermediate decreasing as the percentage of ethanol was increased. The formation mechanism for OHP is proposed.

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The chemical composition and the properties of protein in royal jellies collected from Apis cerana japonica and Apis mellifera were respectively analyzed. A. cerana royal jelly (CRJ) contained more protein and less carbohydrate than A. mellifera royal jelly(MRJ). The water-soluble proteins were analyzed by electrophoresis and immunologically. The major protein components of CRJ and MRJ had different molecular weights, isoelectric points, and immunological characteristics.

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Human or bovine lactoferrin (LF) and lactoferricin^(R) (LFcin), a peptide derived from the N-terminal region of LF, each have the ability to stimulate the release of neutrophil-activating polypeptide interleukin 8 (IL-8) from human polymorphonuclear leukocytes (neutrophils, PMNs). This findinig suggests that LF and LFcin may both function as immunomediators for activating the host defense system. A basic peptide, protamine, exerted the same effect as that of LF and LFcin, suggesting the importance of the basic nature of LF and LFcin in acting as an inducer of IL-8 release from PMNs.

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The substrate speciticity of a p-nitrophenyl α-D-glucopyranosyl-uronic acid-hydrolyzinig enzyme (PNP-GAase) isolated from snail acetone powder has been investigated with various substrates, such as P-nitrophenyl α-D-glucopyranosyluronic acid (PNP-GA), 2-O-α-D-glucopyraniosyluronic acid-D-xylose (GA-2X), 2-O-(4-O-methyl-α-D-glucopyranosyluronic acid)-D-xylose (MeGA-2X), and O-α-D-glucopyranosyluronic acid-α-D-glucopyranosiduronic acid (GA-GA). The K_m (mM) and V_max (μmol of glucuronic acid formed/mg of en-zyme protein/min) toward these substrates were as follows; 0.13 and 3.21 for PNP-GA, 0.33 and 0.089 for GA-2X, 17.6 and 0.094 for MeGA-2X, and 0.36 and 0.015 for GA-GA, respectively. The results indicate that the PNP-GAase specifically hydrolyzed PNP-GA, however, the enzyme had broad substrate specificity.

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The first synthesis of squamostanal-A (1), separated as a degradation product of tetrahydrofuranic acetogenins, is described. Iodide 7, which corresponds to the latent aldehyde moiety of 1, was prepared through a 2-step sequence from 13-[tetrahydropyran-2′-yloxy]-2-tridecyn-1-o1 (5). The NaHMDS-based coupling reaction of 7 with γ-lactone 8 gave compound 9, which by a 3-step sequence, was coverted to 1.

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The effects of in vivo lipopolysaccharide administration on serum lipid metabolism were studied in normal and hepatoma-bearing rats. Changes in serum lipid levels and adipose tissue lipase (lipoprotein lipase and hormone-senisitive lipase) activities following injection of lipopolysaccharide into normal rats resembled those in hepatoma-bearing rats. These results suggest the presence of some common factor(s) involved in the incidence of abnormal lipid metabolism upon lipopolysaccharide injection and hepatoma transplantation.

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Two protein kinases, one from soybeans and the other from rice leaves, were partially purified by sequential chromagography. These protein kinases, which had molecular masses of 47 and 50 kDa, respectively, were found to be activated by calcium and phosphatidylserine and catalyze the phosphorylation of serine residue(s) of histone III-S.

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Enzymatic deamidation of α_s1-casein was done by using Ca²⁺-independent microbial transglutaminase (MTGase) of a variant of Streptoverticillium mobaraense. Although the amount of deamidated glutamine residues in α_s1-casein was not as high as that of the case using guinea pig liver transglutaminase (GTGase), the improvements in pH-solubility and Ca²⁺-sensitivity protile of the substrate protein were comparable to it. To do the enzymatic deamidation without chemical acylation of Lys residues of α_s1-casein, several immobilized MTGase were prepared with two types of chitosan beads. Although neither α^s1-casein nor β-casein was deamidated, dimethyl casein and citraconylated soy 7S globulin were deamidated by using the immobilized enzymes.

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The synthesis of bestatin (1), based on the regio-selective introduction of a nitrogen function to a non-protected diol 9, is described.

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The structure of an asparagine (N-) linked oligosaccharide of pokeweed lectin-B (PL-B) and the amino acid sequences around two glycosylation sites were identified. The pyridylamino (PA) oligosaccharide prepared from PL-B was eluted as a single peak on both reversed-phase (RP-) HPLC and size-fractionation (SF-)HPLC, and its structure was estimated to be Manα1→6(Manα1→3)(Xylβ1→2) Manβ1→4GlcNAcβ1→4(Fucα1→3)GlcNAc by a combination of component analysis, successive exoglycosidase digestions, IS-MS analysis, and 500 MHz ¹H-NMR. Two tryptic glycopeptides were isolated from the reduced and S-pyridylethylated PL-B after gel filtration followed by RP-HPLC, indicating the presence of two glycosylation sites in PL-B. The amino acid sequences around the two glycosylation sites were determined to be Cys-Gly-Val-Asp-Phe-Gly-Asn(CHO)-Arg.

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Biological activities of the carboxy-terminal (C-terminal) deletion mutants of human interleukin-11 (IL-11) were analyzed. Removal of only 1 amino-acid residue (leucine) from the C-terminus caused nearly an 80% loss of its biological activity. This shows the importance of C-terminus of human IL-11 in terms of conserving the biological activity.

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Thermophilic bacteria isolated from spoiled acidic beverages were identified on the basis of DNA-DNA homology and 16S ribosomal DNA sequence similarity to Alicyclobacillus acidoterrestris. This result suggested that the isolates were a new type of spoilage bacterium in acidic beverages in Japan.

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Amylolytic activity that converts soluble starch to α, α-trehalose (trehalose), was found in the cell homogenate of the hyperthermophilic acidophilic archae, Sulfolobus solfataricus KM1. DEAE chromatography of the homogenate as well as other new reliable assay methods showed two enzymes to be essential for this activity. These enzymes, a glycosyltransferase and an amylase, were purified to homogeneity and characterized. Their molecular masses were 76 kDa and 61 kDa and activities were maximal at 70-80°C and 70-85°C, respectively. High thermostability was noted for each. The reaction products by the two enzymes on maltooligosaccharides were identified by ¹H- and ¹³C-NMR spectra and HPLC analysis. The cooperative mechanism of the two enzymes was used in a new enzymatic pathway for trehalose synthesis from starch.

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A well-defined X-ray fiber diffraction pattern was obtained from a stretched film of the calcium salt of a new uronic acid containing polysaccharide designated as beijeran in the deacetylated form, poly[→3)-α-D-GalUA-(1→3)-β-L-Rham-(1→3)-α-D-Glc-(1→]. The oriented film showed no diffraction spots, indicating it to be amorphous. However, when annealed at high temperature, the film exhibited high crystallinity. All the visible reflections could be indexed in terms of a monoclinic unit cell with the following dimensions: a=1.297; b=1.676; c(fiber axis)=2.509nm; and γ=106.50°. The length of the fiber axis and the absence of meridional reflections at any odd layer line indicate that an extended two-fold helical conformation was made up of two trisaccharide residues.

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A molt-inhibitinig hormone (Prc-MIH) was isolated from the sinus glands of the American crayflsh, Procambarus clarkii, and its amino acid sequence was determined. It comprised 75 amino acid residues and had an amidated carboxyl terminus. The amino acid sequence was much more similar to MIH of the shore crab (Cam-MIH) than to MIH of the American lobster (Hoa-MIH).

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