The interaction of distamycin with ColE1 DNA was examined by using differential scanning calorimetry (DSC) taking the helix-coil transition theory of DNA into consideration. Our results here strongly indicate that the affinity of distamycin to DNA, at a low distamycin concentration, depends highly on the DNA sequence, and preferential binding occurs to the sites of four to six successive A-T pairs having two or more successive G-C pairs on both their ends.
Very fast tryptic degradation of the silkworm diapause hormone was found and the degradation pathway was analyzed by moderating the reaction conditions. It proceeded via cleavage at Arg23 and finally at Arg15 of DH. As the C-terminal structure of DH was essential for exhibiting bioactivity, the first cleavage caused rapid inactivation of the hormone. This tryptic digestion was strongly suppressed by adding VAP-map, a synthetic analog of the cuticular peptide of silkmoths, Bm ACP-6.7 (VAP-peptide), which is a natural synergist of DH. VAP-map suppressed the enzymic reaction by interacting with the substrate, but not with the enzyme.
Past research on diapause-inducing substances of the silkworm has isolated an extremely lipophilic peptide and demonstrated its unique characteristics. In the present work, similar lipophilic proteins were searched for in the shell of the shrimp, Pandalus borealis, and one novel protein, Pb CP-12.7, was isolated. Its structure comprising 126 amino acids was revealed by a combination of a sequence analysis and the enzymic fragmentation technique. Pb CP-12.7 is unique in that it was insoluble in neutral-slightly basic water, but highly soluble in some organic solvents. It contained an abundance of hydrophobic amino acids and repeating sequences. In addition, it was adsorbed to chitin, a major component of the shell of the shrimp.
The correlation equation for the concentration-mortality curve of a specific pesticide and a control pesticide was used to adapt the effectiveness data from residual activity tests to the corresponding chemical application concentration, to calculate the constant with the decreasing speed equation, and to calculate the application dose necessary to maintain an equal residual effect period by the same equation. The calculated value is the amount of pesticide needed to perform with the same efficiency as the control pesticide (effectiveness and period of effectiveness), which is vitally important to effectively select an appropriate pesticide.
Incorporation studies administering 2H- and 13C- labeled mevalonate (MVA) and 13C-labeled glucose to suspension cultured cells of the liverwort, Ptychanthus striatus, were carried out in order to examine the biosynthesis of the phytyl side-chain of chlorophyll a. Administration of 13C- and 2H-labeled MVA provided evidence for the involvement of the MVA pathway in the phytyl side-chain biosynthesis and preferential labeling of the farnesyl diphosphate (FPP)-derived portion. An alternate labeling pattern in the phytyl side-chain was observed which was slightly different to the nonequivalent labeling in other liverworts, such as Heteroscyphus planus and Lophocolea heterophylla and in the hornwort, Anthoceros punctatus. The labeling pattern observed after the administration of 13C-labeled glucose revealed the simultaneous involvement of the non-MVA pathway in the phytol biosynthesis of P. striatus cells.
A stereo controlled synthesis of the biologically active neolignan, (+)-dehydrodiconiferyl alcohol (1) was achieved. This synthetic method was also efficient for preparing its enantiomer and other derivatives with biological activity.
Five new lupeol esters, lup-20(29)-ene-3β-yl eicosanoate, docosanoate, tetracosanoate, hexacosanoate and octacosanoate, were isolated as a mixture from the twig bark of Japanese pear (Pyrus serotina Rehd.) cv. Shinko, together with lupeol and epifriedelinol. Their structures were determined by spectral analyses including 2D-NMR experiments.
The drimane sesquiterpenes, (+)-albicanol (2) and (+)-albicanyl acetate (3), were synthesized from an optically active bicyclic diol [(+)−1] that had been obtained via the recently developed optical resolution of a general synthetic intermediate for drimane sesquiterpenes. The crucial step in the previous syntheses was markedly improved by the modified Wittig methylenation of a silyloxy ketone (7). The high overall yield (77% in 4 or 5 steps from (+)-1) by this total synthesis makes it possible to synthesize the other biologically active drimane sesquiterpenes.
Effects of some organic acids and monoterpenes on production of β-thujaplicin were studied in Cupressus lusitanica suspension cultures. The fungal elicitor-induced biosynthesis of β-thujaplicin was promoted by the feedings of malate, pyruvate, fumarate, succinate, and acetate. These results suggest some relationships between acetate/pyruvate metabolism and β-thujaplicin biosynthesis, or between tricarboxylic acid cycle and β-thujaplicin biosynthesis. Feedings of C. lusitanica suspension cultures with some monoterpenes inhibited elicitor-triggered β-thujaplicin biosynthesis, but 2-carene and terpinyl acetate feedings significantly improved the β-thujaplicin production of C. lusitanica suspension cultures. These results indicate a possible involvement of terpinyl acetate and 2-carene in β-thujaplicin biosynthesis, as well as potential uses of these monoterpenes in large-scale β-thujaplicin production.
Glucose isomerase (GI) from Streptomyces olivaceoviridis E-86 is a unique enzyme, very acid-stable with a large potential for corn sweetener industries. The gene encoding this unique enzyme was cloned by a simple two-step PCR method, and expressed in Escherichia coli. A single open reading frame consisting of 1164 base pairs (70.7 mol% of G+C content) that encoded a polypeptide composed of 388 amino acid residues (Mr 42,993) was found. The E. coli transformant carrying the gene overproduced the recombinant GI (rGI) and the enzyme was successfully expressed as a tetramer under the transcriptional control of the tac-promoter. The purified recombinant enzyme was indistinguishable from that of the authentic enzyme e.g. molecular weight, immunological properties, N-terminal amino acid sequences, subunit structures, and temperature and pH profiles. The relationships between structure and properties of the enzymes are also discussed.
Collagen-induced arthritis (CIA) can be induced in DBA/1J mice by immunization with bovine type II collagen (bCII) and is a model of some types of human autoimmune rheumatoid arthritis. In this study we examined whether preimmunization of the mice with various antigens could inhibit the development of CIA. Preimmunization of the mice with an extract of the house dust mite Dermatophagoides farinae (mite antigen), chicken ovalbumin, or keyhole limpet hemocyanin strongly inhibited CIA development, but hen egg lysozyme, β-lactoglobulin from bovine milk or myelin basic protein from guinea pig brain did not substantially affect CIA development. Splenic T cells and serum antibodies specific for mite antigen did not cross-react with bCII. Preimmunization of the mice with mite antigen did not affect the IFN-γ and proliferative response of splenic T cells to bCII, nor serum antibody responses. The most inhibitory constituent had a molecular weight between 1,000 and 10,000.
We describe the preparation of Fab fragments of a humanized anti-human high-affinity IgE receptor (FcεRIα) antibody potentially useful for treatment of IgE-mediated allergic diseases. IgE-binding capacities of sixteen combinations of light and heavy chains of four recombinant anti-FcεRIα antibodies, chimeric CRA2, humanized CRA2, chimeric CRA4, and humanized CRA4, were compared. A combination in which both chains were of humanized CRA2 had the highest activity. Stable transfectant clones of four kinds of host cells expressing recombinant antibodies were established. CHO-K1 cells were the most productive. Serum-free media suitable for culture of the stable CHO-transfectant clones were screened. The concentration of the humanized CRA2, which the most productive clone secreted into the chosen serum-free medium, was approximately 100 μg/ml. A procedure for the purification of the antibody, papain-digestion, and purification of Fab fragments was established. The highly purified humanized Fab fragments are suitable for use to examine their in vivo activity and immunogenicity in primates.
Isocitrate lyase (ICL) and malate synthase (MS) of a psychrophilic marine bacterium, Colwellia maris, were purified to electrophoretically homogeneous state. The molecular mass of the ICL was found to be 240 kDa, composed of four identical subunits of 64.7 kDa. MS was a dimeric enzyme composed of 76.3 kDa subunits. N-Terminal amino acid sequences of the ICL and MS were analyzed. Purified ICL had its maximum activity at 20°C and was rapidly inactivated at the temperatures above 30°C, but the optimum temperature for the activity of MS was 45°C. NaCl was found to protect ICL from heat inactivation above 30°C, but the salt did not stabilize MS. Effects of temperatures on the kinetic parameters of both the enzymes were examined. The Km for the substrate (isocitrate) of ICL was decreased with decreasing temperature. On the other hand, the Km for the substrate (glyoxylate) of MS was increased with decreasing temperature. The calculated value of free energy of activation of ICL was on the same level as that of MS.
During previous work on deriving inosine-producing mutants of Escherichia coli, we observed that an excess of adenine added to the culture medium was quickly converted to hypoxanthine. This phenomenon was still apparent after disruption of the known adenosine deaminase gene (add) on the E. coli chromosome, suggesting that, like Bacillus subtilis, E. coli has an adenine deaminase. As the yicP gene of E. coli shares about 35% identity with the B. subtilis adenine deaminase gene (ade), we cloned yicP from the E. coli genome and developed a strain that overexpressed its product. The enzyme was purified from a cell extract of E. coli harboring a plasmid containing the cloned yicP gene, and had significant adenine deaminase [EC 188.8.131.52] activity. It was deduced to be a homodimer, each subunit having a molecular mass of 60 kDa. The enzyme required manganese ions as a cofactor, and adenine was its only substrate. Its optimum pH was 6.5-7.0 and its optimum temperature was 60°C. The apparent Km for adenine was 0.8 mM.
Cyclin B cDNA was cloned from the ovary of the starfish Asterina pectinifera and analyzed by RT-PCR and 3'- and 5'-RACE techniques. The cDNA consists of a 0.13-kb upstream untranslated region, a 1.22-kb coding region, and a 0.86-kb downstream untranslated region. The open reading frame encoded a polypeptide of 404 amino acid residues with a calcutated molecular weight of 45,692. All the characteristic sequences, such as destruction and cyclin boxes, cyclin B motif, and cytoplasmic retention and nuclear export signals, were found in the newly cloned cyclin B cDNA. The deduced amino acid sequence of the cyclin B cDNA was highly homologous in the middle and carboxy terminal regions to that from mature eggs of the same organism, but quite different in the amino terminal region. Evidence was obtained which suggested that this cyclin B is expressed in immature and maturing oocytes and is the same as that cloned from mature eggs.
Developing seeds of the kidney bean (Phaseolus vulgaris L.) contain several isoforms of starch branching enzymes. Two of them, KBE1 and KBE2, which are the major forms in the premature seeds, were purified as a single band of protein on SDS-PAGE and native PAGE by chromatographies on DEAE-Sepharose, Bio-Gel P-200, and amylose-binding Sepharose 6B. The enzymes had similar pH optimum (7.0), pH stability (7.0-9.5), temperature optimum (25-30°C), and temperature stability (up to 40°C). Additionally, both were inhibited by various divalent metal ions and activated by citrate. Finally, though their N-terminal amino acid sequences were identical, their molecular masses and affinities for amylose differed; 80 kDa and 1.27 mM for KBE1 and 77 kDa and 0.74 mM for KBE2.
Fusobacterium K-60, a ginsenoside Rb1-metabolizing bacterium, was isolated from human intestinal feces. From this Fusodobacterium K-60, a ginsenoside Rb1-metabolizing enzyme, β-glucosidase, has been purified. The enzyme was purified to apparent homogeneity by a combination of butyl-Toyopearl, hydroxyapatite ultragel, Q-Sepharose, and Sephacryl S-300 HR column chromatographies with a final specific activity of 1.52 μmol/min/mg. It had optimal activity at pH 7.0 and 40°C. The molecular mass of this purified enzyme was 320 kDa, with 4 identical subunits (80 kDa). The purified enzyme activity was inhibited by Ba++, Fe++, and some agents that modify cysteine residues. This enzyme strongly hydrolyzed sophorose, followed by p-nitrophenyl β-D-glucopyranoside, esculin, and ginsenoside Rb1. However, this enzyme did not change 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol (IH-901) to 20(S)-protopanaxadiol, while it weakly changed ginsenoside Rb1 to IH-901. These findings suggest that the Fusobacterial β-glucosidase is a novel enzyme transforming ginsenoside Rb1.
A rice bran 57-kDa protein was isolated by affinity chromatography with fibronectin immobilized on agarose. This fibronectin-binding protein designated as RB-57 had an amino-terminal amino acid sequence identical with that of a putative mature form of rice hydroxyproline-rich glycoprotein. A distinct feature of the amino acid composition of RB-57 was the high contents of hydroxyproline and proline representing about 45% of the total amino acids. The sugar analysis indicated that arabinose represented 46.8% of the total carbohydrates. RB-57 showed cell adhesion activity for murine Lewis lung carcinoma cells. The result suggests that RB-57 may play a role in plant cell adhesion, although cell adhesion-promoting activity for plant cells remains to be tested.
Two new β-xylosyl derivatives of ginsenoside Re, 20(S)-protopanaxatriol 6-O-α-L-rhamnopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→4)]-β-D-glucopyranosyl-20-O-β-D-glucopyranoside and 20(S)-protopanaxatriol 6-O-α-L-rhamnopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→6)]-β-D-glucopyranosyl-20-O-β-D-glucopyranoside, were respectively synthesized from p-nitrophenyl β-D-xylopyranoside and phenyl β-D-xylopyranoside as donors and ginsenoside Re as the acceptor in 25% acetone and acetonitrile by a cellulase preparation from Trichoderma viride and a β-galactosidase preparation from Aspergillus oryzae. The latter enzyme preparation also catalyzed the hydrolysis of ginsenoside Re to the minor saponin, ginsenoside Rg2.
We have adapted a fluorescence in situ hybridization (FISH) method to detect nascent RNA molecules of the imprinted SNRPN gene at the initial transcription sites in nuclei of human HL60 and WI38 cells. Simultaneous detection of RNA and DNA of SNRPN by FISH using the cosmid probe confirmed its monoallelic expression in these cell lines. Treatment of both cells by inhibitors of DNA methylation and histone deacetylation resulted in time-dependent increase of the cell population with the biallelic expression of SNRPN.
Galβ-(1→3)-GalNAc-linked hexapeptide was synthe-sized by a transglycosylation using Galβ-(1→3)-Gal-NAcβ-pNP as a donor and a serine-containing hexapeptide as an acceptor using endo GalNAc-ase from Streptomyces sp.. The Galβ-(1→3)-GalNAc residue was transferred to the hydroxyl group of the serine residue of the peptide. The total yield of the glycopeptide via this process was better than that of the chemoenzymatic method. This process was confirmed to be a versatile method for the synthesis of O-linked glycopeptides.
Effects of ICRF-193, a topoisomerase II inhibitor, on metaphase chromosome preparations were examined. A short-time exposure of this drug to human HL60 cells in a suspension culture before harvest resulted in obtaining more extended metaphase chromosomes. The length of chromosome 6 identified by fluorescence in situ hybridization was twice as long with this drug treatment. Together with effectiveness for adherent HepG2 cells, these results suggest that treatments with ICRF-193 provide a simple and reliable method for extended metaphase chromosome preparations from cultured cells.
Immunosuppressants isolated from Streptomyces filipinensis and S. hygroscopicus were identified with pentalenolactone I and hygromycin A, respectively. The compounds as well as cyclosporin A showed immunosuppressant activity in the mixed lymphocyte reaction, and pentalenolactone I and cyclosporin A suppressed IL-2 production, however, hygromycin A did not. Hygromycin A may have immunosuppressant activity by a different mechanism from pentalenolactone I, cyclosporin A and tacrolimus.
Autodigestion of subtilisin ALP I (ALP I), secreted from the alkalophilic Bacillus sp. NKS-21 and its predicted amino acid sequence having about 60% identity with other alkaline subtilisins, was examined under alkaline conditions. At the alkaline pH of 12, ALP I was rapidly degraded, and almost no breakdown products were detectable. However, by incubating ALP I at 5°C for an extended time, a couple of specific peptides (26.7 kDa and 25.6 kDa) were accumulated. Each of them was purified and amino acid sequences of these fragments were found. Both peptides appeared to start at Gly-19 of the mature sequence of ALP I.
We isolated the ipdc gene coding for indole-3-pyruvic acid decarboxylase (IPDC), a key enzyme in the indole-3-pyruvic acid pathway for indole-3-acetic acid biosynthesis, in the plant growth-promoting rhizobacterium Azospirillum lipoferum FS. Gel mobility-shift assay showed the presence of two DNA-binding proteins that might be involved in regulation of the ipdc gene expression.
The expression of pathogenesis-related protein-5d (PR-5d) and ethylene-responsive transcriptional factors (ERF1-4) for basic PR-proteins was measured in cultured tobacco cells. The PR-5d transcript markedly increased with culture, but only constant levels of transcripts of ERF1 (trans-activator) and ERF3 (repressor) were detected in cells. These different responses of PR-5d and ERF genes were also observed under NaCl stress; PR-5d transcript decreased and ERF levels were constant under NaCl stress. The effect of post-transcriptional regulation of ERF activity, especially ERF3, on the expression of basic PR genes including PR-5d is discussed.
Dietary proteins are recognized by the gastrointestinal tract to display physiological functions, however, the sensory mechanism of the intestinal mucosa is not known. We examined binding properties between the rat small intestinal brush-border membrane (BBM) and proteins by using a surface plasmon resonance biosensor. BBM and solubilized BBM prepared from the rat jejunum bound to casein immobilized on the sensor surface, but not to bovine serum albumin. The ileal BBM showed less binding to casein than the jejunal BBM. Solubilized BBM binding to immobilized α-casein was slightly inhibited by aminopeptidase inhibitors, but still more inhibited by addition of casein with the inhibitors. Guanidinated casein inhibited the solubilized BBM binding to α-casein more strongly than casein (casein sodium and α-casein) inhibited. Trypsinization of solubilized BBM abolished its binding activity to α-casein. These results indicate that some membrane protein, but not aminopeptidases, contained in BBM interacts with dietary proteins, and that guanidinated casein has a higher affinity for BBM than intact casein. These binding intensities for proteins were closely correlated to physiological responsiveness, and are possibly involved in a sensory system for dietary protein in the intestine.
Cut lettuce stored at 4°C gradually turned brown on the cut section after several days of storage. Three factors for enzymatic browning, the polyphenol content, polyphenol oxidase activity, and phenylalanine ammonia-lyase (PAL) activity, were examined during the cold storage of cut lettuce. A relationship between the browning and PAL activity was apparent. We tried to prevent this browning by using the two enzyme inhibitors, 2-aminoindane-2-phosphonic acid (AIP), an inhibitor of the phenylpropanoid pathway, and glyphosate, an inhibitor of the shikimate pathway. AIP and glyphosate significantly inhibited the browning of cut lettuce. The polyphenol content and PAL activity were both reduced by the treatment with AIP. These results show that regulating the biosynthesis of polyphenols is essential to prevent the browning of cut lettuce.
This study examined the effects of soybean β-conglycinin, from which phytate was mostly removed, on the plasma lipids in young and adult rats. Male Wistar young (6 week-old) and adult (21 week-old) rats were fed high cholesterol diets containing 20% casein, soy protein isolate (SPI), or soybean β-conglycinin for 10 days. In young rats, although the food intake of the β-conglycinin group was higher than those of the casein and SPI groups, the weight gain was significantly lower than those of the other groups. However, in adult rats, the weight gain was not different among the groups. In young and adult rats, relative liver weights of SPI and β-conglycinin groups were significantly lower than that of the casein group, and the degree of the reduction was more marked in the β-conglycinin group than in the SPI group. In young rats, the plasma triglyceride level was significantly lower in the SPI and β-conglycinin groups than that in the casein group. In addition, the plasma triglyceride level of the β-conglycinin group was significantly lower than that of the SPI group. Plasma total cholesterol levels of the SPI and β-conglycinin groups were significantly lower than that of the casein group. However, there was little difference in the lowering effect between SPI and β-conglycinin. These results indicate that soybean β-conglycinin may have lowering functions not only on plasma total cholesterol level, but aiso on plasma triglyceride level.
The production of digested bacterial cell powder (DBCP) as a source of reduced-form folates for pigs was studied. Trimethoprim-resistant mutants of Brevibacterium lactofermentum ATCC 13869 accumulated a significantly higher amount of the reduced form of folate in the cells than the wild-type strain. DBCPs were prepared from the resistant mutant strain and the wild-type strain. The utilization of the reduced-form of folate in DBCP was evaluated by measuring the plasma folate level after orally administering DBCP to Göttingen minipigs. The folates in both DBCPs proved to have equally high bioavailability in the pigs.
Although several studies have shown that milk protein components have a wide range of biological activities, the potential role of these proteins in the gastrointestinal mucosal defense system is less well elucidated. In this study, we investigated the effect of the major proteins in cow’s milk on gastric mucosal injury by using two acute ulcer models in Wistar rats. Gastric mucosal injury was induced by either intragastric 60% ethanol-HCl or water-immersion restraint stress (23°C, 7 h). Each test milk protein was orally administered 30 min before the induction of gastric injury. Among the major milk proteins, α-lactalbumin (α-LA) is demonstrated to have a marked protective effect against ethanol-induced gastric injury, with the same potency as that of the typical antiulcer agent, Selbex. Whey protein isolate (WPI), which contained 25% α-LA, also protected against gastric injury, while casein showed no effect. Comparative studies on the protective effect of the four major components of WPI, β-lactoglobulin, α-LA, bovine serum albumin and γ-globulins (immunoglobulins), on the basis of their contents in WPI revealed that α-LA was responsible for the protective effect of WPI, being about 4-fold more effective than WPI itself. α-LA showed dose-dependent protection against gastric injury induced by stress as well as ethanol. Pretreatment with indomethacin (10 mg/kg body weight, s.c.), which is a potent inhibitor of endogenous prostaglandin synthesis, resulted in a significant reduction in the protective effect of α-LA. These results indicate that α-LA has marked antiulcer activity as an active component of cow’s milk protein, and suggest that α-LA intake may serve to protect against gastric mucosal injury, in part through endogenous prostaglandin synthesis.
We compared the antioxidative activities of seven hydrocoumarins with those of α-tocopherol for the oxidation of tetralin and linoleic acid in a homogeneous solution. Hydrocoumarins exhibited a higher induction period than that of α-Toc in both systems. However, the rate of oxygen absorption during the induction period for α-Toc was slower than that of the hydrocoumarins in both systems. In addition, 6,7-dihydroxy-4,4-dimethylhydrocoumarin showed less cytotoxicity toward human fibroblasts than did 2,6-di-t-butyl-4-methylphenol.
We previously demonstrated that a methionine-threonine-supplemented low (8.5%) casein diet (8.5CMT) reduced symptoms such as proteinuria in nephritic rats without severe protein malnutrition. In this study, we examined whether or not L-arginine supplementation to 8.5CMT would exacerbate proteinuria and other symptoms in nephritic rats. Male Wistar rats with glomerulonephritis induced by a single intravenous injection of nephrotoxic serum were fed either a 20% casein diet (control), 8.5% casein diet, 8.5CMT, or L-arginine-supplemented 8.5CMT (8.5CMTA) for 16 days. The 8.5CMTA, as compared with the 8.5CMT, aggravated proteinuria and glomerulonephritis. Administration of L-NG-nitroarginine methyl ester, an inhibitor of nitric oxide synthase, to 8.5CMTA-fed nephritic rats by drinking water for 14 days canceled the adverse effect of L-arginine on proteinuria and histopathological damage in glomeruli. These results suggest that the supplementation of L-arginine makes exacerbation via nitric oxide production in glomerulonephritis.
Oral administration of a protein without adjuvant brings about oral tolerance (systemic hyporesponsiveness) to that protein by mechanisms such as antigen-induced apoptosis. We monitored the number and apoptosis induction of CD4+ T cells in antigen-specific T cell receptor transgenic mice fed the antigen ovalbumin to identify where events leading to oral tolerance occurred. The antigen was distributed throughout the body, causing apoptosis and a decrease in cell number of CD4+ T cells in most of the lymphoid system: the spleen, peripheral lymph nodes, and the thymus which was not previously reported to be affected. Although apoptosis was induced in the Peyer’s patches, the cell number did not change. Unexpectedly, T cells in the mesenteric lymph nodes did not undergo apoptosis; instead, they were more numerous as compared to that in the case of control animals not administered the antigen. The results suggested that the orally administered antigen activated the intestinal immune system, while it induced immune tolerance in other sites
Two different phosphopeptide (PPP) fragments derived from partially dephosphorylated hen egg yolk phosvitin were prepared by tryptic digestion, and their Ca2+ binding property compared with that of commercial casein phosphopeptides (CPP). The smaller fragment of less than 1 kDa and O-phospho-l-serine did not bind Ca2+ to any significant extend, while PPP of 1-3 kDa showed a higher ability than CPP to render soluble calcium. The results show that not only the phosphoserine residues are critical for Ca2+ binding, but also the molecular size of the phosphopeptides.
LPLPR, a complement C3a agonist peptide, with hypocholesterolemic activity was introduced into the homologous site of soybean proglycinin A1aB1b subunit by site-directed mutagenesis. This modified proglycinin was expressed in E. coli and recovered from the insoluble fraction. LPLPR was released by the action of chymotrypsin and trypsin as expected. Furthermore, two peptides (RPSYLPLPR and PSYLPLPR) with extended sequence at the amino-terminus of LPLPR were obtained. Their ileum-contracting activity was 9 ∼ 13 times stronger than that of LPLPR. The overall yields of purified LPLPR, RPSYLPLPR and PSYLPLPR were 25%, 12%, and 0.7% respectively.
Glycitein, as one of the three major isoflavones in soybeans, directly but significantly (about 50%) suppressed the proliferation of MC3T3-E1 and stimulated bone-related protein (alkaline phosphatase (ALP) and osteocalcin (OC)) expression. These results indicate that glycitein suppresses the proliferation of osteoblasts and promotes differentiation from its progenitor.
We compared the lipophilicity and toxicity of the four flavonols, galangin, kaempferol, quercetin and myrice-tin, which respectively have no, one, two and three hydroxyl groups on the B-ring. The lipophilicity was in the order of myricetin < quercetin < kaempferol < galan-gin. The cytotoxicity determined by a colony-formation assay with Chinese hamster lung fibroblast V79 cells was in the order of quercetin < kaempferol < galangin<myricetin. Apart from myricetin, the order of lipophilicity was the same as that of cytotoxicity, implying that the cytotoxicity was attributable to the lipophilicity. The cytotoxicity of myricetin was attributable to the hydrogen peroxide formed by autoxidation.
Ceotrichum sp. FO347-2 could use refined sardine oil as a sole carbon source. Dry cell mass reached a maximum of 0.788 g per g of the oil added for 72 h. Total weight of the cellular lipids was largest around 24 h, when the contents of triglyceride and free fatty acid were 63.6 and 22.2%, respectively. Docosahexaenoic acid (DHA) was incorporated and concentrated in the cellular lipids, and the content reached 25.9% for 24 h, adding sardine oil containing 12.3% DHA. DHA and eicosapentaenoic acid were also concentrated in residual lipids outside the cells. Using tuna head oil containing 26.8% DHA, FO347-2 was compared with Candida guilliermondii FO726A with respect to DHA incorporation. FO347-2 and FO726A accumulated similar amounts of DHA, i.e. 53 and 55 mg, respectively, in 1 g of freeze-dried cells after 24-h cultivation at 30°C. The recovery rates of DHA from the tuna oil for FO347-2 and FO726A were 19.4 and 19.7%, respectively.
Aspartokinase III (AKIII), one of three isozymes of Escherichia coli K-12, is inhibited allosterically by L-lysine. This enzyme is encoded by the lysC gene and has 449 amino acid residues. We analyzed the feedback inhibition site of AKIII by generating various lysC mutants in a plasmid vector. These mutants conferred resistance to L-lysine and/or an L-lysine analogue on their host.The inhibitory effects of L-lysine on and heat tolerance of 14 mutant enzymes were examined and DNA sequencing showed that the types of mutants were 12. Two hot spots, amino acid residue positions 318-325 and 345-352, were detected in the C-terminal region of AKIII and these enzyme regions may be important in L-lysine-mediated feedback inhibition of AKIII. Feedback resistant lysC relieved on L-threonine hyper-producing strain, B-3996, from reduced L-threonine productivity by addition of L-lysine, and furthermore increased L-threonin productivity even when no addition of L-lysine. It suggested that the bottleneck of L-threonine production of B-3996 was AK and feedback resistant lysC was effective because of the strict inhibition by cytoplasmic L-lysine.
Carboxypeptidase Y (CPY) has been used as a maker enzyme for investigations on intracellular transport of vacuolar proteins and on vacuolar biogenesis in Saccharomyces cerevisiae. We describe the cloning and characterization of the CPY homologue encoding gene (cpyA) from the filamentous fungus Aspergillus nidulans. The cpyA gene has one intron and encodes a protein with 552 amino acids containing a putative signal sequence and pro-sequence. The predicted CpyA protein is highly similar in sequence with carboxypeptidases from several yeast species and contains a catalytic triad (Asp-His-Ser) like that of serine carboxypeptidase. The cpyA disruptant cells showed reduced levels of intracellular carboxypeptidase. These results suggest that the cpyA gene encodes a vacuolar carboxypeptidase in A. nidulans.
Microbial degraders of poly(3-hydroxybutyrate) (PHB) were isolated from soil. Arthrobacter sp. strain W6 used not only PHB as a carbon source, but also PHAs such as poly(3-hydroxybutyrate-co-[5%]3-hydroxyvalerate, poly(3-hydroxybutyrate-co-[14%]3-hydroxyvalerate), and poly(3-hydroxybutyrate-co-[22%]3-hydroxyvalerate). PHB-depolymerase was purified to homogeneity from the culture broth of Arthrobacter sp. strain W6 by a procedure involving DEAE- and butyl-Toyopearl column chromatographies. The Mr of the enzyme was estimated to be about 47,000 by SDS-polyacrylamide gel electrophoresis. The enzyme was most active at pH 8.5 and 50°C, and was inhibited by phenylmethylsulfonyl fluoride, Hg2+, Ag+, and Pb2+.
Aurantiamide acetate was isolated from the fermentation broth of Aspergillus penicilloides for the first time. Aurantiamide acetate inhibited cysteine proteinases, in particular, cathepsin L (184.108.40.206) and B (220.127.116.11) with IC50 of 12 μM and 49 μM, respectiveiy. In the adjuvant-arthritic rat model, subcutaneously administered 10 mg/kg body weight of this compound suppressed hind paw swelling.
A novel denitrifier Alcaligenes sp. STC1 was identified. The strain efficiently denitrifies under an atmosphere of 10% oxygen (O2) where Paracoccus denitrificans, one of the most studied aerobic denitrifiers, had less denitrifying activity, indicating that the strain has an O2-torelant denitrifying system. It denitrified by using C1-carbon sources such as formate and methanol as well as glucose, glycerol, and succinate. The genes for the copper-containing nitrite reductase and azurin of this C1-using denitrifier were cloned. Their predicted products of them were similar to those of their counterparts and the maximum similarities were 90% and 92%, respectively.
A new method to estimate the number of polyhydroxyalkanoates (PHA)-degraders in soil and to isolate degraders, called the film-MPN method, is proposed. The incubation time was measured by the first order reaction (FOR) model. This method was used to estimate numbers of poly (3-hydroxybutyrate-co-3-hydroxyvalerate)[P(3HB-co-3HV)]- and poly(3-hydroxyvalerate-co-4-hydroxybutyrate)[P(3HB-co-4HB)]-degraders in garden soil (4.30 × 105 and 2.15 × 105 aerobic degraders per gram of dry soil, respectively). The number of P(3HB-co-3HV)-degraders in paddy field soil was 5.06 × 105 aerobic degraders per gram dry soil. Also, several P(3HB-co-3HV)-degraders were isolated directly from positive-growth tubes of high dilution.
Polymerase chain reaction (PCR) for the flagellin central domain coding region (FCD-PCR) was applied to the detection and discrimination of Bacillus subtilis DB9011, a strain with useful functions in agriculture. Cross-reactions were observed in 4 B. subtilis strains with similar flagellin genes (hag). Alignment of partial amino-acid sequences of flagellin and the results of PCR for the 16S/23S rRNA spacer in 11 B. subtilis strains suggested the presence of a group including strains with antifungal activity (DB9011 and others).
We tried some improvement of inosine production using an inosine-producing mutant of Escherichia coli which is deficient in purF (phosphoribosyl-pyrophosphate (PRPP) amidotransferase gene), purA (succinyl-adenosine 5'-monophosphate (AMP) syn-thetase gene), deoD (purine nucleoside phosphorylase gene), purR (purine repressor gene) and add (adenosine deaminase gene), and harboring the desensitized PRPP amidotransferase gene as a plasmid. The guaB (inosine 5'-monophosphate (IMP) dehydrogenase gene) disruption brought about a slightly positive effect on the inosine productivity. Alternatively, the gsk (guanosine-inosine kinase gene) disruption caused a considerable amount of guanosine accumulation together with a slight increase in the inosine productivity. The further addition of guaC (guanosine 5'-monophosphate (GMP) reductase gene) disruption did not lead to an increased guanosine accumulation, but brought about the decrease of inosine accumulation.
The biocatalytic stereoselective hydrolysis of 2-hydroxy-3-nitropropionic acid esters was studied. Forty enzymes and three hundred microorganism strains were examined for their ability to hydrolyze ethyl 2-hydroxy-3-nitropropionic acid. Nocardia globerula IFO13150 gave n-butyl (R)-2-hydroxy-3-nitropropionate with a 92% enantiomeric excess (ee) and the corresponding carboxylic acid with a 92%ee, which was easily converted to (S)-isoserine, a useful β-amino acid.