We have previously isolated and characterized the rice (Oryza sativa) cDNAs, OsCyc1/OsCPS4, OsCyc2/OsCPS2, OsKS4, OsDTC1/OsKS7, OsDTC2/OsKS8 and OsKS10, which encode cyclases that are responsible for diterpene phytoalexin biosynthesis. Among the other members of this gene family, OsCPS1 and OsKS1 have been suggested as being responsible for gibberellin biosynthesis, OsKSL11 has recently been shown to encode stemodene synthase, and the functions of the three other diterpene cyclase genes in the rice genome, OsKS3, OsKS5 and OsKS6, have not yet been determined. In this study, we show that recombinant OsKS5 and OsKS6 expressed in E. coli converted ent-copalyl diphosphate into ent-pimara-8(14),15-diene and ent-kaur-15-ene, respectively. Neither product is a hydrocarbon precursor required in the biosynthesis of either gibberellins or phytoalexins. OsKS3 may be a pseudogene from which the translated product is a truncated enzyme. These results suggest that the diterpene cyclase genes responsible for gibberellin and phytoalexin biosynthesis are not functionally redundant.
Sauriols A and B belong to a class of diarylbutane-lignans and exhibit antifeedant activity. We succeeded in the first synthesis of sauriols A and B by using a simple and efficient asymmetric dimerization of a cinnamic acid derivative as the key step.
Two anthraquinone derivatives, named anthrasesamones D and E, were isolated from the roots of Sesamum indicum. Their respective structures were determined to be 1,2,4-trihydroxy-3-(4-methylpent-3-enyl)anthraquinone and 1,2-dihydroxy-3-(4-methylpent-3-enyl)anthraquinone on the basis of spectroscopic evidence.
CI-8 is the chymotrypsin inhibitor in hemolymph from the silkworm, Bombyx mori. It occurs in the midgut at the spinning stage of larva, but little information on the mechanism of its uptake in the midgut is available. We found that two polypeptides interacting with CI-8 are in the midgut membrane, and we purified them using a biotinylated CI-8, viz., p29 and p60, having molecular sizes of 29 kDa and 60 kDa respectively. The structures of p29 and p60 were examined by N-terminal amino acid sequencing and peptide mass mapping, including tryptic digestion. p29 was highly similar to the matured 19G1-30K lipoprotein from hemolymph, but p60 was novel. Purified p29 was recognized by anti-19G1-30K antibody, and was confirmed to be similar to 19G1-30K. The antibody also neutralized the CI-8 binding ability of p29 in the midgut membrane. p29 and p60 are perhaps proteinaceous factors involved in the uptake of CI-8 into the midgut through the membrane.
The Synechococcus sp. PCC7942 strain carrying a missense mutation in the peptide-binding domain of DnaK3, one of the essential dnaK gene products, revealed temperature-sensitive growth. We also isolated suppressor mutants of this strain. One of the suppressors was mapped in the ribosomal protein gene rpl24 (syc1876), which encodes the 50S ribosomal protein L24. Subcellular localization of three DnaK proteins was determined, and the results indicated that a quantity of DnaK3 was dislocated from membrane-bound polysomes when dnaK3 temperature-sensitive mutant was incubated at non-permissive temperatures. Furthermore, we examined the photosystem II reaction center protein D1 and detected a translational intermediate polypeptide in membrane-bound polysome fractions prepared from dnaK3 temperature-sensitive cells grown at high temperature. These characteristic features of DnaK3 localizations and detection of D1 protein intermediate were not observed in the suppressor mutant even at high temperatures.
The mode of action of a potent elicitor, N-acetylchitooligosaccharide, in rice plants was examined. In intact seedlings, no significant uptake of the elicitor via the roots was observed within 3 h, whereas rapid uptake was observed in excised leaves. Rapid and transient expression of an elicitor-responsive gene, EL2, was induced in the leaves of intact seedlings sprayed with the elicitor or in the roots and leaves of intact seedlings by immersing roots in the elicitor solution. Histochemical analysis indicated that EL2 was expressed in cells exposed to the elicitor of root and leaves. In seedlings treated with the elicitor for 1 d or longer, hyphal growth of rice blast fungus was significantly delayed, and an accumulation of auto-fluorescence around the infection site was observed. Two defense-related genes, PR-1 and PR-10 (PBZ1), were induced in a systemic and local manner by elicitor treatment, in correlation with the induction of resistance against rice blast fungus. N-Acetylchitoheptaose did not inhibit the hyphal growth of the fungi. These results indicate the occurrence of systemic signal transmission from N-acetylchitooligosaccharide in rice plants.
The product of an open reading frame (ORF) (called YdbR) identified while analyzing the Bacillus subtilis genome has been classified as an Asp-Glu-Ala-Asp (DEAD) protein, but the biological function and enzymology of YdbR have not been characterized in detail. Here we show that recombinant YdbR-His6 purified from Escherichia coli is an ATP-independent RNA binding protein. It also possesses RNA-dependent ATPase activity stimulated not only by total RNA from B. subtilis but also by an RNA that is irrelevant to that of B. subtilis. Functional analysis indicated that the growth rate of a ΔydbR mutant strain of B. subtilis was reduced as compared with that of the wild type not only at 37 °C, but more severely at 22 °C.
In order to save energy during the pulp making process, we tried to use white-rot basidiomycete, Trametes hirsuta, which degrades lignin efficiently. But a decrease in paper strength caused by cellulolytic activity ruled this out for practical application. Since the cellulolytic activity of the fungus must be decreased, we purified and characterized a cellobiose dehydrogenase (CDH) that was reported to damage pulp fiber. The CDH in the culture filtrate of C. hirsutus was purified by freeze-thawing and chromatographic methods. The pI of the enzyme was 4.2 and its molecular weight was 92 kDa. The optimal temperature and pH of the enzyme were 60–70 °C and 5.0 respectively. Since the purified CDH decreased the viscosity of pulp in the presence of Fe(III) and cellobiose, it was shown that the suppression of CDH should be an effective way to reduce cellulose damage.
The present investigation showed that active processes were involved in the uptake of 2,4-dichlorophenoxyacetate (2,4-D) by Delftia acidovorans MC1. With 2,4-D-grown cells, uptake at pH 6.8 was highly affine and showed a complex pattern-forming intermediary plateau at 20–100 μM 2,4-D. The kinetics became increasingly sigmoidal with raising of the pH to 7.5 and 8.5, and complexity disappeared. The apparent maximum was obtained at around 400 μM 2,4-D at either pH, and amounted to 15–20 nmol/min*mg protein. Higher substrate concentrations resulted in significant inhibition. With cells grown on (RS)-2-(2,4-dichlorophenoxy)propionate, 2,4-D uptake increased significantly and reached 45 nmol/min*mg, hinting at induction of a specific carrier(s). The kinetic characteristics made it apparent that several proteins contribute to 2,4-D uptake in MC1. An open reading frame was detected which has similarity to genes encoding major facilitator superfamily (MFS) transporters. Mutant strains that lacked this gene showed altered kinetics with decreased affinity to 2,4-D at pH 6.8. A mutant with complete deficiency in phenoxyalkanoate utilization showed an almost linear uptake pattern hinting at sole diffusion. Cloning of tfdK encoding a specific transporter for 2,4-D resulted in an increased uptake rate and, above all, higher affinity at slightly alkaline conditions due to hyperbolic kinetics. The presence of carbonylcyanide m-chlorophenylhydrazone led to the subsequent strong inhibition of 2,4-D uptake, suggesting proton symport as the likely active mechanism.
Azoreductases have been characterized as enzymes that can decolorize azo dyes by reducing azo groups. In this study, genes encoding proteins having homology with theazoreductase gene of Bacillus sp. OY1-2 were obtained from Bacillus subtilis ATCC6633, B. subtilis ISW1214, and Geobacillus stearotherophilus IFO13737 by polymerase chain reaction. All three genes encoded proteins with 174 amino acids. The deduced amino acid sequences of azoreductase homologs from B. subtilis ISW1214, B. subtilis ATCC6633, and G. stearotherophilus IFO13737 showed similarity of 53.3, 53.9, and 53.3% respectively to that of Bacillus sp. OY1-2. All three genes were expressed in Escherichia coli, and were characterized as having the decolorizing activity of azo dyes in a β-NADPH dependent manner. The transformation of several azo dyes into colorless compounds by recombinant enzymes was demonstrated to have distinct substrate specificity from that of azoreductase from Bacillus sp. OY1-2.
The genes for a nitric oxide reductase-like cytochrome bc complex were cloned from a thermophilic, chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6. The structural genes norC and norB, which encode cytochrome c and cytochrome b subunits of the complex respectively, are probably transcribed as a tricistronic operon with a following gene encoding a putative membrane protein. NorC has, unusually, two hydrophobic transmembrane spans in its N-terminus. Immunoblot analysis showed that expression of NorC was induced by nitrate, nitrite, or sodium nitropurusside, suggesting that the norCB gene product is a denitrification enzyme, nitric oxide reductase. The consensus sequences for the DNR/NnrR-type or the NorR/FhpR-type nitric oxide-sensing regulators of proteobacteria were not found in the norC promoter region, but consensus −35 and −10 sequences were found in this region. These results indicate that strain TK-6 has a nitrogen oxide-sensing regulatory system that differs from proteobacterial systems.
The copper stimulon in Escherichia coli consists of four regulons, the CueR-, CusS/CusR-, CpxA/CpxR-, and YedV/YedW regulons. E. coli mutants defective in cpxRA showed higher sensitivity to copper than the wild type. A total of 15 promoters were found to be induced in E. coli culture upon exposure to copper in a CpxA/CpxR-dependent manner. After gel-shift and DNase I foot-printing analyses, a conserved tandem repeat of pentanucleotide sequence, GTAAA(N)4–8GTAAA, with a conserved A of 4-bp upstream of each pentamer, was identified to be the CpxR-binding site. The difference in the orientation and location of the CpxR box is discussed with respect to the regulation mechanism among CpxR-regulon genes.
Chitinase [EC 188.8.131.52] is an enzyme that can hydrolyze the β-1,4 linkage between N-acetyl-D-glucosamine in chitin. In the genome database of the hyperthermophilic archaeon Pyrococcus furiosus, we found two adjacent genes (PF1233 and PF1234) homologous to those of the chitinase of Thermococcus kodakaraensis. In the cultured medium of P. furiosus, however, no chitinase activity was detected. On analysis of the structural gene of P. furiosus, it appears that one nucleotide insertion in PF1234 caused a frame shift and separated a gene. By deletion of one nucleotide in PF1234, the best match was achieved between chitinases of T. kodakaraenesis and P. furiosus. We succeeded in constructing an artificial recombinant chitinase exhibiting hydrolytic activity toward not only colloidal but also crystalline chitins at high temperature. Furthermore, by analyzing the characteristics of the domains, a recombinant enzyme comprising two domains exhibiting high activity toward crystalline chitin was prepared.
Double-stranded RNA-activated protein kinase (PKR) plays an important role in interferon-induced antiviral responses, and is also involved in intracellular signaling pathways, including the apoptosis, proliferation, and transcription pathways. In this study, a new 120-kDa PKR-associated protein designated double-stranded RNA binding protein (DRBP)-120 was identified using co-immunoprecipitation with anti-PKR antiserum and two-dimensional electrophoresis. Furthermore, DRBP-120 is a double-stranded RNA (dsRNA)-binding protein, and it was detected in both the cytoplasm and the nucleus of HeLa cells associated with PKR.
An Arabidopsis thaliana cDNA library was introduced into a Saccharomyces cerevisiae mutant that lacks ScBOR1 (YNL275W), a boron (B) efflux transporter. Five cDNAs were identified that confer tolerance to high boric acid. The nucleotide sequence analysis identified the clones as a polyadenylate-binding protein, AtPAB2; a ribosomal small subunit protein, AtRPS20B; an RNA-binding protein, AtRBP47c′; and two Myb transcription factors, AtMYB13 and AtMYB68. The expression of these five genes also conferred boric acid tolerance on wild-type yeast. Two yeast genes, ScRPS20 and ScHRB1, that are similar to the isolated clones, were necessary for this boric acid tolerance. The possible roles of these A. thaliana and S. cerevisiae genes in boric acid tolerance are discussed.
Plant growth retardants (PGRs) reduce the shoot growth of plants by inhibiting gibberellin biosynthesis. In this study, we performed detailed analyses of the inhibitory effects of PGRs on Arabidopsis abscisic acid (ABA) 8′-hydroxylase, a major ABA catabolic enzyme, recently identified as CYP707As. In an in vitro assay with CYP707A3 microsomes expressed in insect cells, uniconazole-P inhibited CYP707A3 activity more effectively than paclobutrazol or tetcyclacis, whereas the other PGRs tested did not inhibit it significantly. Uniconazole-P was found to be a strong competitive inhibitor (Ki=8.0 nM) of ABA 8′-hydroxylase. Uniconazole-P-treated Arabidopsis plants showed enhanced drought tolerance. In uniconazole-P-treated plants, endogenous ABA levels increased 2-fold as compared with the control, and co-application of GA4 did not suppress the effects, indicating that the effects were not due to gibberellin deficiency. Thus uniconazole-P effectively inhibits ABA catabolism in Arabidopsis plants. We also discuss the structure-activity relationship of the azole-type compounds on ABA 8′-hydroxylase inhibitory activity.
The moc1 gene in Schizosaccharomyces pombe was found as to overcome sterility caused by high expression of adenylyl cyclase. The moc1 gene was found to be identical with sds23 and psp1. Although psp1 has been reported to be essential for growth, sds23 has not been. To clarify this apparent discrepancy, we first assessed independently the phenotypes of the moc1 disruptant. We confirmed that the deletion mutant of moc1 is sterile, sensitive to high salt, and grows slowly at higher and lower temperatures, and that mutant cells are elongated. Besides these phenotypes, we found that viability of the moc1 disruptant was rapidly lost at the stationary phase. We confirmed that the Moc1 protein is phosphorylated in the stationary phase and also under nitrogen-starved conditions. We examined the significance of this phosphorylation of Moc1 by creating the S333A or S333D mutant Moc1. Interestingly, while S333D mutant Moc1 is lower in inducing sexual development, S333A mutant Moc1 is higher in this than the wild type, suggesting that phosphorylation of Moc1 affects sexual development. The other phenotypes, such as sensitivity to high salt and higher temperature and elongation of cells, were not affected by mutation of S333A nor S333D. We found that Moc1-GFP localized to both the cytosol and the nucleus during mitotic growth, but accumulated in the nucleus in mating cells and then enriched in spores, and that this localization shift was negatively regulated by the cAMP pathway. This and the observations above suggest that nuclear localized Moc1 is an inducer of sexual development. Thus, in addition to the roles of moc1/sds23/psp1 in mitosis and stress response, it is also important for the survival and sexual development of fission yeast, but phosphorylation of Moc1 only affects the sexual development.
We have previously shown a significant decrease in the ethylene production in tomato fruit from the RIN/rin genotype. In this present study, we evaluated the amount of 1-aminocyclopropane-1-carboxylic acid (ACC) and the gene expression and enzymatic activities of ACC synthase (ACS) and ACC oxidase (ACO) to find which type of regulation influenced this low ethylene production. The results suggest that the decreased ethylene production was due to transcriptional regulation of the ACS and ACO genes by the heterozygous effect of the rin gene.
The 1,6-β-D-glucanase gene (neg1) of Neurospora crassa was disrupted by repeat-induced point mutations. Sequence analysis of the neg1 gene in the R12-1 mutant showed that 9 nucleotides within the coding region of the gene changed from GC to AT. The base transition of C to A at position 662 resulted in a codon. No apparent phenotypic changes were observed in the mutant, but Congo-red, SDS, and cetyltrimethyl ammonium bromide (CTAB), which affect fungal cell walls or membranes, markedly inhibited the hyphal growth of the mutant at a concentration that does not inhibit growth in the wild type.
Transgenic Arabidopsis thaliana plants carrying a single copy of integrated DNA can be identified by single-step genomic polymerase chain reaction. The reaction employs two sets of primer pairs with the same melting temperature that amplify the amplicons derived from the integrated T-DNA together with those from an endogenous single-copy gene as a reference. When the band intensity ratio is one, this means that the transgenic plants are carrying a single copy of the integrated gene per haploid.
Previously we reported the purification of soluble γ-glutamyltransferases (GGTs) from radish cotyledon. Subcellular fractionation of radish cells revealed that soluble GGT is a vacuolar enzyme. Acivicin, a GGT inhibitor, mediated the in vivo catabolism inhibition of the glutathione S-conjugate generated from endogenous glutathione and exogenously supplied monochlorobimane. Thus soluble GGT is possibly involved in the catabolism of glutathione S-conjugates.
The pgsBCA-gene disruptant from Bacillus subtilis ISW1214, i.e., MA41, does not produce poly-γ-glutamate (PGA). We newly constructed an MA41 recombinant bearing the plasmid-borne PGA synthetic system, in which PGA production was strictly controlled by the use of xylose. Unlike the parent strain, ISW1214, the genetically engineered strain produced abundant PGA in both L-glutamate-rich and D-glutamate-rich media.
A differential display was performed to analyze differential gene expression in the brains of mice in association with dietary high beef-tallow. Consumption of a high beef-tallow diet downregulated the expression of ADP-ribosylation factor-like protein 8B (Arl8B) mRNA in the brain. Arl8B mRNA was widely expressed in the mouse brain, including primary neuronal cells. The current study indicates that green fluorescent protein-fused Arl8B protein accumulated at the growth cones in primary neuronal cells, and that protrusions of human embryonic kidney 293 (HEK293) cells were significantly elongated by overexpression of Arl8B, suggesting an important role of Arl8B in neurite formation.
We examined the effect of administering an isoflavone-rich fermented soybean extract (FSBE) on the serum cholesterol concentrations in male rats and in intact and ovariectomized (OVX) female rats. Dietary FSBE decreased the serum cholesterol concentrations in intact female and OVX rats, but did not affect the concentrations in male rats. Dietary FSBE increased the hepatic total and esterified cholesterol contents in the intact female rats, but decreased them in the OVX rats. This hypocholesterolemic effect was not a simple estrogenic effect because it has appeared in some reports that estrogen administration decreased serum cholesterol both male and female rats. Dietary FSBE increased the hepatic low-density lipoprotein receptor (LDLR) gene expression in the intact female rats as has previously been reported from many studies, but did not affect that of the OVX rats. Further investigation is needed into the hypocholesterolemic mechanism of FSBE.
A novel technique is reported for softening plant tissues while retaining their shape by impregnating them with macerating enzymes under reduced pressure after defrosting the frozen plants. Samples were removed immediately from the enzyme solution after the freeze-infusion treatment, and the hardness was measured. Six enzymes and three enzymes were respectively chosen from 18 commercial enzymes for softening burdock roots and bamboo shoots. The tissue degradation due to impregnation of the tissues with the enzymes and the reaction time were investigated. Burdock roots and bamboo shoots were progressively softened during the reaction: the hardness reached 1.0×104 N/m2 or less. The water-soluble dietary fiber contents increased as a result of the freeze-infusion treatment. This softening technique, which retained the food shape, could enhance the production of food products for elderly persons and those under nursing care. Foods produced by this method can replace current minced and liquid dietary components.
We surveyed the contamination of 315 Japanese- and western-style desserts and 247 human hands by Staphylococcus aureus and other staphylococcal bacteria. The most frequently isolated staphylococcal bacterium was S. warneri, followed by S. aureus. Only 1.9% of western-style desserts were contaminated by S. aureus strains, while 19.4% and 13.0% of Japanese-style desserts and human hands respectively were contaminated. Ninety-four isolates of S. aureus were characterized as to their biological properties and enterotoxigenicity. Although staphylococcal enterotoxins (SEs) were detected by enzyme-linked immunosorbent assay in the cultured broth of all S. aureus isolates, the reversed passive latex agglutination method and the polymerase chain reaction showed only 39 (41.5%) and 40 (42.6%) samples respectively as SE-positive. The predominant type of SE was SEB (67.5%), and eight strains produced SEA. None of the S. aureus strains had penicillin-binding protein 2′, showing that methicillin-resistant S. aureus was not present in the samples.
The near-infrared (NIR) spectra of such metals as Cu(II), Mn(II), Zn(II) and Fe(III) in HNO3 in the 700–1860 nm region were subjected to a partial least-squares regression analysis and leave-out cross-validation to develop chemometric models. The models yielded a coefficient of determination in cross validation of 0.9744 [Cu(II)], 0.9631 [Mn(II)], 0.9154 [Zn(II)] and 0.741 [Fe(III)]. The regression coefficients for Cu(II), Mn(II) and Zn(II), but not for Fe(III), showed strong negative peaks at around 1050–1200 nm, a zone where spectral bands have been reported to decrease with increasing pH value. A positive peak at around 710–750 nm, which may have been due to water absorption, was observed in regression coefficients of Cu(II), Mn(II) and Zn(II) but not in Fe(III), while a negative peak was observed in that for Fe(III) at around 710–750 nm. These results indicate that the divalent cations [Cu(II), Mn(II) and Zn(II)] showed different absorption in the NIR region from the trivalent cation [Fe(III)], suggesting that the vibration mode of water, which mirrors the interaction between cations and water, may be influenced by valency.
Acrylamide (AAm) is produced in food through the reaction of asparagine and reducing sugar. We examined several methods of reducing the level of AAm using potato tubers. The fried model system that we employed consisted of thin slices that were first treated in water under different conditions before frying. A sufficient amount of water present in the fry material acts as an inhibitor against the formation of AAm and allows only a negligible amount of AAm to form. It was found that given the low content of water, the fry material temperature was sufficiently high to allow a relatively large level of AAm to form. Examination of water treatment prior to frying revealed that higher-temperature treatment water and longer treatment time resulted in the formation of lower levels of AAm. Moreover, removing some of the residual heat had an inhibiting effect on the formation of AAm.
To clarify the alternative mechanisms to vitamin E (VE) regulating lipid peroxide accumulation in the liver after docosahexaenoic acid (DHA) ingestion, we examined the relationship between the DHA-induced lipid peroxide formation and induction of the xenobiotic transporters, Ral-binding GTPase-activating protein (RalBP1) and multidrug resistance-associated proteins 1, 2 and 3 (MRP1–3), in the liver of rats fed with DHA. The test diets contained DHA and linoleic acid (LA) (8.7% and 2.1% of total energy, respectively) with different levels of dietary VE (normal and low: 68 and 7.7 mg of alpha-tocopherol equivalent per kg diet, respectively), and the control diet contained LA alone (11.5% of total energy). The rats were fed with these experimental diets for 14 d. The proportions of DHA in the liver, kidney and heart were higher in the DHA-fed groups than in the LA-fed group. The tissue thiobarbituric acid values as an index of lipid peroxidation were also significantly higher in the DHA-fed groups, but the value did not differ between the DHA-fed groups with different VE levels. In the liver, there were no significant differences in the glutathione S-transferase (GST) and aldehyde dehydrogenase (ALDH) activities or in the expression of GST M2, RalBP1, MRP1 and MRP2 mRNA. However, the obvious induction of expression of liver MRP3 mRNA and tendency to produce the protein were recognized after DHA ingestion. This study is the first to report the gene expression of MRP3 by DHA ingestion. There might exist, therefore, some relationship between the DHA intake and MRP3 induction in regulating lipid peroxide accumulation in the liver.
Cranberry, which is rich in polyphenols, including anthocyanins and proanthocyanidins, has been found to have various effects beneficial to human health, including prevention of urinary tract infections. These effects have been associated with polyphenols in the fruit. We investigated the excretion of anthocyanins in human urine after ingestion of cranberry juice. Eleven healthy volunteers consumed 200 ml of cranberry juice containing 650.8 μg total anthocyanins. Urine samples were collected within 24 h before and after consumption. Six of 12 anthocyanins identified in cranberry were quantified in human urine by HPLC coupled with electrospray ionization and tandem mass spectrometry (HPLC–ESI–MS–MS). Among these, peonidin 3-O-galactoside, the second most plentiful anthocyanin in the juice, was found most abundantly in urine within 24 h, corresponding to 41.5 nmol (56.1% of total anthocyanins). The urinary levels of anthocyanins reached a maximum between 3 and 6 h after ingestion, and the recovery of total anthocyanins in the urine over 24 h was estimated to be 5.0% of the amount consumed. This study found high absorption and excretion of cranberry anthocyanins in human urine.
The retention of deoxynivalenol in noodles and bread made from naturally-contaminated flour was examined by a chemical analysis (HPLC) and bioassays. The retention level of deoxynivalenol obtained from both assays was reduced by boiling process, although only the bioassays showed it to have been reduced by baking. This study is the first to estimate the exposure to deoxynivalenol from the consumption of the final products of wheat flour in Japan.
Chitosan interaction with soybean β-conglycinin β3 was investigated by thermal unfolding experiments using CD spectroscopy. The negative ellipticity of the protein was enhanced with rising solution temperature. The transition temperature of thermal unfolding of the protein (Tm) was 63.4 °C at pH 3.0 (0.15 M KCl). When chitosan was added to the protein solution, the Tm value was elevated by 7.7 °C, whereas the Tm elevation upon addition of chitosan hexamer (GlcN)6 was 2.2 °C. These carbohydrates appear to interact with the protein stabilizing the protein structure, and the interaction ability could be evaluated from the Tm elevation. Similar experiments were conducted at various pHs from 2.0 to 3.5, and the Tm elevation was found to be enhanced in the higher pH region. We conclude that chitosan interacts with β-conglycinin through electrostatic interactions between the positive charges of the chitosan polysaccharide and the negative charges of the protein surface.
It was found that Acidithiobacillus thiooxidans has sulfite:ubiquinone oxidoreductase and ubiquinol oxidase activities in the cells. Ubiquinol oxidase was purified from plasma membranes of strain NB1-3 in a nearly homogeneous state. A purified enzyme showed absorption peaks at 419 and 595 nm in the oxidized form and at 442 and 605 nm in the reduced form. Pyridine ferrohaemochrome prepared from the enzyme showed an α-peak characteristic of haem a at 587 nm, indicating that the enzyme contains haem a as a component. The CO difference spectrum of ubiquinol oxidase showed two peaks at 428 nm and 595 nm, and a trough at 446 nm, suggesting the existence of an aa3-type cytochrome in the enzyme. Ubiquinol oxidase was composed of three subunits with apparent molecular masses of 57 kDa, 34 kDa, and 23 kDa. The optimum pH and temperature for ubiquinol oxidation were pH 6.0 and 30 °C. The activity was completely inhibited by sodium cyanide at 1.0 mM. In contrast, the activity was inhibited weakly by antimycin A1 and myxothiazol, which are inhibitors of mitochondrial bc1 complex. Quinone analog 2-heptyl-4-hydoroxyquinoline N-oxide (HOQNO) strongly inhibited ubiquinol oxidase activity. Nickel and tungstate (0.1 mM), which are used as a bacteriostatic agent for A. thiooxidans-dependent concrete corrosion, inhibited ubiquinol oxidase activity 100 and 70% respectively.
Lactobacillus reuteri is one of the dominant lactobacilli found in the gastrointestinal tract of various animals. A surface protein of L. reuteri 104R, mucus adhesion promoting protein (MapA), is considered to be an adhesion factor of this strain. We investigated the relation between MapA and adhesion of L. reuteri to human intestinal (Caco-2) cells. Quantitative analysis of the adhesion of L. reuteri strains to Caco-2 cells showed that various L. reuteri strains bind not only to mucus but also to intestinal epithelial cells. In addition, purified MapA bound to Caco-2 cells, and this binding inhibited the adhesion of L. reuteri in a concentration-dependent manner. Based on these observations, the adhesion of L. reuteri appears due to the binding of MapA to receptor-like molecules on Caco-2 cells. Further, far-western analysis indicated the existence of multiple receptor-like molecules in Caco-2 cells.
S-layer homology (SLH) module polypeptides were derived from Clostridium thermocellum S-layer proteins Slp1 and Slp2 and cellulosome anchoring protein AncA as rSlp1-SLH, rSlp2-SLH, and rAncA-SLH respectively. Their binding specificities were investigated using C. thermocellum cell-wall preparations. rAncA-SLH associated with native peptidoglycan-containing sacculi from C. thermocellum, including both peptidoglycan and secondary cell wall polymers (SCWP), but not to hydrofluoric acid-extracted peptidoglycan-containing sacculi (HF-EPCS) lacking SCWPs, suggesting that SCWPs are responsible for binding with SLH modules of AncA. On the other hand, rSlp1-SLH and rSlp2-SLH associated with HF-EPCS, suggesting that these polypeptides had an affinity for peptidoglycan. A binding assay using a peptidoglycan fraction prepared from Escherichia coli cells definitely confirmed that rSlp1-SLH and rSlp2-SLH specifically interacted with peptidoglycan but not with SCWP.
2-Deoxy-scyllo-inosose (DOI) synthase is the enzyme participating in biosynthesis of 2-deoxystreptamine (DOS)-containing aminoglycoside antibiotics. The gene which encodes the enzyme can be a marker for screening of DOS-containing aminoglycoside-producer and exploration of its biosynthetic gene. Further, this enzyme is expected to be of use in industry, because it converts sugar into 6-membered carbocycle. In the present study, we identified 21 clones encoding DOI synthase from environmental DNA by degenerate PCR. They were clearly divided into two groups. One appeared to derive from actinomycetes, and the other from non-actinomycetes. The latter group was larger (17 clones) than the former (four clones) despite the fact that only one strain of non-actinomycete was identified for DOS-containing aminoglycoside production. This result indicates that there are still many unidentified non-actinomycetes for DOS-containing aminoglycoside biosynthesis. We showed the possibility of identification of novel aminoglycoside-producing non-actinomycete from soil, and for development of more efficient enzymes for industrial use.
A culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of Schizophyllum commune has an activity to form protoplasts from S. commune mycelia, and a combination of α-1,3-glucanase and chitinase I, which were isolated from the filtrate, brings about the protoplast-forming activity. The gene of α-1,3-glucanase was cloned from B. circulans KA-304. It consists of 3,879 nucleotides, which encodes 1,293 amino acids including a putative signal peptide (31 amino acid residues), and the molecular weight of α-1,3-glucanase without the putative signal peptide was calculated to be 132,184. The deduced amino acid sequence of α-1,3-glucanase of B. circulans KA-304 showed approximately 80% similarity to that of mutanase (α-1,3-glucanase) of Bacillus sp. RM1, but no significant similarity to those of fungal mutanases. The recombinant α-1,3-glucanase was expressed in Escherichia coli Rosetta-gami B (DE 3), and significant α-1,3-glucanase activity was detected in the cell-free extract of the organism treated with isopropyl-β-D-thiogalactopyranoside. The recombinant α-1,3-glucanase showed protoplast-forming activity when the enzyme was combined with chitinase I.
We characterized a trifluoroleucine-resistant mutant of Saccharomyces cerevisiae, TFL20, that has a mutation in the LEU4 gene. We monitored the concentration of extracellular i-AmOH and intracellular amino acids, and compared the ratios of gene expression in TFL20 with the wild-type strain, K30. We found that the LEU1, LEU2, and BAT1 genes were up-regulated in TFL20 for metabolism, and that TFL20 simultaneously produced as much i-AmOH and leucine as K30 does.