Filamentous fungi are frequently used for the production of industrial enzymes, since they produce a variety of enzymes including polysaccharide-degrading enzymes. Among the many filamentous fungi, Aspergillus species, such as A. oryzae and A. niger, are known as strong producers of amylolytic enzymes. We have been studying on the regulatory mechanisms underlying the expression of A. oryzae amylolytic genes. Based on analyses using a hybrid model system of A. nidulans transformed by a gene encoding A. oryzae Taka-amylase A, the major amylase (taaG2), we have found that three factors, CCAAT-box binding protein, CreA, and AmyR, are involved in taaG2 gene expression and regulation. In this review, the focus is on the CCAAT-box binding protein of filamentous fungi. The assembly, nuclear translocation, and transcriptional enhancement mechanisms of the CCAAT-box binding protein are discussed.
Generally, when microbes assimilate macromolecules, they incorporate low-molecular-weight products derived from macromolecules through the actions of extracellular degrading enzymes. However, a Gram-negative bacterium, Sphingomonas sp. A1, has a smart biosystem for the import and depolymerization of macromolecules. The bacterial cells directly incorporate a macromolecule, alginate, into the cytoplasm through a “superchannel”, as we named it. The superchannel consists of a pit on the cell surface, alginate-binding proteins in the periplasm, and an ATP-binding cassette transporter in the inner membrane. Cytoplasmic polysaccharide lyases depolymerize alginate into the constituent monosaccharides. Other than the proteins characterized so far, novel proteins (e.g., flagellin homologs) have been found to be crucial for the import and depolymerization of alginate through genomics- and proteomics-based identification, thus indicating that the biosystem is precisely constructed and regulated by diverse proteins. In this review, we focus on the structure and function of the bacterial biosystem together with the evolution of related proteins.
Buckwheat often causes severe allergic reactions, even when its ingestion level is extremely low. Therefore, buckwheat is listed in several countries as a common food allergen. In addition to common buckwheat and Tartarian buckwheat that are cultivated and consumed widely, wild buckwheat may be potentially allergenic. Food containing undeclared buckwheat poses a risk to patients with the buckwheat allergy. We describe in this report a PCR method to detect buckwheat DNA by using primers corresponding to the internal transcribed spacer region and the 5.8S rRNA gene. The method is buckwheat-specific and compatible with both cultivated and wild buckwheat of the Fagopyrum spp. Its sensitivity was sufficient to detect 1 ppm (w/w) of buckwheat DNA spiked in wheat DNA. This method should benefit food manufacturers, clinical doctors, and allergic patients by providing information on the presence of buckwheat contamination in food.
Prodrug-oriented molecular design was attempted for the potent acyclic neonicotinoid insecticide, clothianidin (1-(2-chloro-5-thiazolylmethyl)-3-methyl-2-nitroguanidine). Molecules bearing a CH2COCH2 bridge linking the 1,3-NH ends of clothianidin or their acetals would possibly be hydrolyzed, regenerating the mother compounds. This strategy was used to prepare seven acetals of clothianidin-based molecules that combined 2-chloro-5-thiazolylmethyl, 6-chloro-3-pyridylmethyl or 3-tetrahydrofurfuryl with a nitroimine, cyanoimine or nitromethylene group. The key intermediate, 1,3-diamino-2,2-dimethoxypropane, was prepared from the dihydroxyacetone dimer in four steps. A selected acetal showed a characteristic nerve-impulse pattern for neonicotinoids on an excised American cockroach ganglion, although the neuroblocking activity was fairly low. Some acetals were highly insecticidal against the pea aphid at 0.8–20 ppm 7 days after a spray treatment, this being in a contrast to their far weaker activity by injection into American cockroaches. The biological results suggest that the intrinsic insecticidal activities of the acetals are weak, but would exhibit enhanced activity if hydrolyzed in an external environment.
The mechanism of floral scent emission was studied in Petunia axillaris, a plant with a diurnal rhythm of scent output. The emission rate of each volatile compound oscillated in synchrony with its endogenous concentration, so that the intensity of the floral scent appeared to be determined by the endogenous concentrations. The composition of major volatiles in the flower tissue and the flower headspace showed characteristic differences. A negative correlation was found between the boiling points of the volatile compounds and the ratio of their emitted and endogenous concentrations, indicating that the composition of the floral scent depends directly on the endogenous composition of the volatile compounds. We conclude that in P. axillaris, the physiological regulation of floral scent emission operates not in the vaporization process but in the control of the endogenous concentrations of volatiles through biosynthesis and metabolic conversion.
A method for quantifying indole-3-acetic acid (IAA) and its conjugates with the six amino acids, Ala, -Asp, -Ile, -Glu, -Phe and -Val, in rice (Oryza sativa) by using high-performance liquid chromatography coupled with electrospray ionization and tandem mass spectrometry (HPLC–ESI–MS/MS) is described. Samples from the rice plant or callus were treated with 80% acetone in water containing 2.5 mM diethyl dithiocarbamate. Each extract was partially purified in C18 cartridge column for solid-phase extraction (SPE) and subjected to HPLC–ESI–MS/MS without converting the product. The detection limit was 3.8 fmol for IAA, and 0.4–2.9 fmol for the IAA amino acid conjugates. The method was applied to the analysis of IAA and its conjugates in rice seedlings, dehulled rice and calli, using 20–100 mg tissue samples.
The acylation of the rare sugar, D-allose (the C-3 epimer of D-glucose), with fatty acid vinyl esters was successfully carried out using Candida antarctica lipase in acetonitrile at 45 °C to give D-allose 6-alkanoates with high regioselectivity in good yields.
From a mixture of N-acetylglucosaminyl-β-cyclodextrin (GlcNAc-βCD) and lactose, β-D-galactosyl-GlcNAc-βCD (Gal-GlcNAc-βCD) was synthesized by the transfer action of β-galactosidase. GlcNAc-maltotriose (Glc3) and Gal-GlcNAc-Glc3 were produced with hydrolysis of GlcNAc-βCD by cyclodextrin glycosyltransferase, and Gal-GlcNAc-βCD by bacterial saccharifying α-amylase respectively. Finally, GlcNAc-Glc3-βCD and Gal-GlcNAc-Glc3-βCD were synthesized in 5.2% and 3.5% yield when Klebsiella pneumoniae pullulanase was incubated with the mixture of GlcNAc-Glc3 and βCD, or Gal-GlcNAc-Glc3 and βCD respectively. The structures of GlcNAc-Glc3-βCD and Gal-GlcNAc-Glc3-βCD were analyzed by FAB-MS and NMR spectroscopy and identified as 6-O-α-(63-O-β-D-N-acetylglucosaminyl-maltotriosyl)-βCD, and 6-O-α-(4-O-β-D-galactopyranosyl-63-O-β-D-N-acetylglucosaminyl-maltotriosyl)-βCD respectively.
The recombinant ovalbumin (OVA) produced in yeast Pichia pastoris was purified from the culture medium by anion exchange chromatography, and its structural characteristics were compared with those of hen egg OVA, mainly from the point of view of posttranslational modification. The expressed OVA consisted of two molecular species immmunoreactive with antibody for hen egg OVA. The two molecular species, 45 and 47 kDa in molecular size, were thought to correspond to mono-glycosylated form and di-glycosylated form respectively. The non-glycosylated form was not produced in the system. The other posttranslational modifications (N-terminal acetylation and phosphorylation) observed in hen egg OVA were not detected in either of the molecular species. The two recombinant proteins displayed almost exactly the same circular dichroism and intrinsic tryptophan fluorescence spectra as hen egg OVA. The melting temperature, Tm, which was determined from the thermal unfolding curve, was almost identical in the two recombinant proteins, despite the difference in glycosylation levels, while it decreased by about 2.5 °C as compared with that of hen egg OVA (77.3 °C). These data indicate that the additional glycosylation to Asn-311 in the recombinant protein does not affect protein conformation or thermostability.
The chloroplastic isoform of monodehydroascorbate (MDA) radical reductase was purified from spinach chloroplasts and leaves. The cDNA of chloroplastic MDA reductase was cloned, and its deduced amino acid sequence, consisting of 497 residues, showed high homology with those of putative organellar MDA reductases deduced from cDNAs of several plants. The amino acid sequence of the amino terminal of the purified enzyme suggested that the chloroplastic enzyme has a transit peptide consisting of 53 residues. A southern blot analysis suggested the occurrence of a gene encoding another isoform homologous to the chloroplastic isoform in spinach. The recombinant enzyme was highly expressed in Eschericia coli using the cDNA, and purified to a homogeneous state with high specific activity. The enzyme properties of the chloroplastic isoform are presented in comparison with those of the cytosolic form.
A protein that binds specifically to the promoter region of chiA was purified from the cell lysate of Streptomyces lividans by using an affinity purification method. Determining the amino-terminal amino acid sequence of the purified protein led to cloning of a gene (cpb1) encoding the chitinase promoter-binding protein, Cpb1. The deduced amino acid sequence of Cpb1 showed significant similarity to the sequences of a group of hypothetical proteins of S. coelicolor that have been revealed by the genome project, and the amino-terminal region of Cpb1 showed similarity to the DNA-binding domains of several transcription factors. The Cpb1 proteins expressed in S. lividans or Escherichia coli showed specific binding activity to the chiA promoter. The disruption of cpb1 resulted in partial relief of the glucose repression of chitinase production, indicating that cpb1 took part in the regulation of chitinase expression in S. lividans.
Royal jelly (RJ) is known to have a variety of biological activities toward various types of cells and tissues of animal models, but nothing is known about its effect on brain functions. Hence, we examined the effect of oral administration of RJ on the mRNA expression of various neurotrophic factors, their receptors, and neural cell markers in the mouse brain. Our results revealed that RJ selectively facilitates the mRNA expression of glial cell line-derived neurotrophic factor (GDNF), a potent neurotrophic factor acting in the brain, and neurofilament H, a specific marker predominantly found in neuronal axons, in the adult mouse hippocampus. These observations suggest that RJ shows neurotrophic effects on the mature brain via stimulation of GDNF production, and that enhanced expression of neurofilament H mRNA is involved in events subsequently caused by GDNF. RJ may play neurotrophic and/or neuroprotective roles in the adult brain through GDNF.
Isopropylmalate dehydrogenase (IPMDH) is an enzyme in the leucine biosynthetic pathway. We isolated three IPMDH ORF sequences from Arabidopsis thaliana, and genes corresponding to these ORF sequences were designated AtIMD1, AtIMD2, and AtIMD3. Deduced amino acid sequences of the three genes contain a putative transit-peptide for plastidic localization. AtIMD1, AtIMD2, and AtIMD3 were able to complement a leu2 mutant of yeast, suggesting that these genes encode functional IPMDH. RT-PCR analysis revealed different tissue specificity of transcript accumulation for the three genes.
Three chitinases, designated gazyumaru latex chitinase (GLx Chi)-A, -B, and -C, were purified from the latex of gazyumaru (Ficus microcarpa). GLx Chi-A,-B, and -C are an acidic class III (33 kDa, pI 4.0), a basic class I (32 kDa, pI 9.3), and a basic class II chitinase (27 kDa, pI>10) respectively. GLx Chi-A did not exhibit any antifungal activity. At low ionic strength, GLx Chi-C exhibited strong antifungal activity, to a similar extent as GLx Chi-B. The antifungal activity of GLx Chi-C became weaker with increasing ionic strength, whereas that of GLx Chi-B became slightly stronger. GLx Chi-B and -C bound to the fungal cell-walls at low ionic strength, and then GLx Chi-C was dissociated from them by an escalation of ionic strength, but this was not the case for GLx Chi-B. The chitin-binding activity of GLx Chi-B was enhanced by increasing ionic strength. These results suggest that the chitin-binding domain of basic class I chitinase binds to the chitin in fungal cell walls by hydrophobic interaction and assists the antifungal action of the chitinase.
Recently we found that firefly luciferase is a bifunctional enzyme, catalyzing not only the luminescence reaction but also long-chain fatty acyl-CoA synthesis. Further, the gene product of CG6178 (CG6178), an ortholog of firefly luciferase in Drosophila melanogaster, was found to be a long-chain fatty acyl-CoA synthetase and dose not function as a luciferase. We investigated the substrate specificities of firefly luciferase and CG6178 as an acyl-CoA synthetase utilizing a series of carboxylic acids. The results indicate that these enzymes synthesize acyl-CoA efficiently from various saturated medium-chain fatty acids. Lauric acid is the most suitable substrate for these enzymes, and the product of lauroyl CoA was identified with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Phylogenetic analysis indicated that firefly luciferase and CG6178 genes belong to the group of plant 4-coumarate:CoA ligases, and not to the group of medium- and long-chain fatty acyl-CoA synthetases in mammals. These results suggest that insects have a novel type of fatty acyl-CoA synthetase.
This paper reports a preparation method for silk sericin hydrogel using the Sericin-hope silkworm, whose cocoons consist almost exclusively of sericin. Sericin solution, prepared from Sericin-hope cocoons, contains intact sericin and forms elastic hydrogels with the addition of ethanol. The sericin hydrogel can be prepared without crosslinking by chemicals or irradiation and might be usable as a naturally occurring biomaterial.
Here we report on the production of functional recombinant SBPase of Chlamydomonas sp. W80 in Escherichia coli and the one-step purification of a polyhistidine-tagged fusion protein. The polyclonal antibody was raised against purified recombinant enzyme and cross-reacted with crude SBPase from Chlamydomonas, spinach, tobacco, and Arabidopsis leaves. Further, we investigated the levels of protein and activity of SBPase in different tissues of Arabidopsis plants.
We examined the tissue distribution, synthesis stage, and ethylene induction of three types of pineapple chitinase using chitinase activity gel and immunoblot analysis. Type A (acidic class III) exists in all tissues, while type B (weakly basic class I, which has strong antifungal activity) and type C (acidic class I) are localized mainly in the leaf and stem. In a pericarp, type A exists at all stages during fruit development, while type B and type C exist only at the early stage. Synthesis of type A is induced by ethylene, while that of types B and C is not affected by it. These results suggest that the physiological roles of these three types of chitinase in pineapple are different.
Deletion of PDR5 gene (Δpdr5) in Saccharomyces cerevisiae led to increased resistance to calcium. The cellular Ca2+ level in the presence of high calcium as estimated by reporter assay in Δpdr5 cells was significantly lower than that in wild-type cells. Membrane Pdr5p levels diminished rapidly during incubation with high calcium in a manner dependent on calcineurin and Pep4p, suggesting a feedback regulatory mechanism for Pdr5p abundance.
Alix/AIP1 is a multifunctional adaptor protein involved in endocytosis, cell adhesion, and cell death. By yeast two-hybrid screening we identified a novel Alix/AIP1-interacting protein named Rab GTPase-activating protein-like protein (RabGAPLP). Interaction between Alix and RabGAPLP was confirmed by pull-down assays using fusion proteins of either glutathione-S-transferase (GST) or chitin-binding domain (CBD) and lysates of cultured mammalian cells expressing the respective proteins. Partial colocalization of FLAG-tagged RabGAPLP and green fluorescent protein (GFP)-fused Alix was observed at cell edges and filopodia-like structures by fluorescence confocal laser scanning microscopic analysis. The identity of RabGAPLP to merlin-associated protein (MAP), one of the interacting partners of neurofibromatosis type 2 (NF2) tumor suppressor gene product (merlin), implies cross-talk of membrane traffic and cell adhesion.
The inhibitory effects of myricitrin on the oxidation of human low-density lipoprotein were investigated before and after its degradation by simulated digestion. Myricitrin strongly inhibited the low-density lipoprotein oxidation induced by either 2,2′-azobis (2-amidinopropane) dihydrochloride or CuSO4 in a concentration-dependent manner. Myricitrin was very stable under an acidic condition (pH 1.8) corresponding to the gastric environment, but it was easily degraded under an alkaline condition (pH 8.5) corresponding to the intestinal environment. However, degraded myricitrin also had a strong inhibitory effect on the oxidative degradation of α-tocopherol, cholesterol and apolipoprotein B-100 in low-density lipoprotein. Our study revealed that myricitrin was degraded into many components under a mildly alkaline condition, but the degraded myricitrin still retained the free radical-scavenging and copper-chelating activities toward low-density lipoprotein.
The cellular components involved in the hypocholesterolemic activity of Kluyveromyces marxianus YIT 8292 were examined in rats fed on a high-cholesterol diet. Whole cells (KM) were heated at 115 °C for 10 minutes and fractionated into water-soluble extract 1 and the insoluble residue (KM-CW). After mechanical disruption by glass beads, KM-CW was separated into the cell wall (KM-W) and water-soluble extract 2. Plasma total cholesterol was decreased by feeding KM-CW or KM-W, but was not changed by feeding extract 1 or extract 2. Feeding KM-CW and KM-W increased the fecal sterol excretion and concentration of short-chain fatty acids (SCFA) in the cecum. The hypocholesterolemic activity of KM-CW was completely abolished by the enzymatic degradation of α-mannan and β-glucan. These results suggest that α-mannan and β-glucan were the major active components of KM, and that its hypocholesterolemic activity may be attributable to the increasing fecal sterol excretion and/or production of SCFA.
A double-blind comparative study was conducted on cedar pollinosis patients in order to evaluate the treatment efficacy of apple polyphenol (Ap). Ap was administered (500 mg) once daily for 12 weeks, starting about 2 weeks prior to cedar pollen dispersion. Pollinosis symptoms during the study were evaluated according to the classification in the guidelines for allergic rhinitis diagnosis and treatment. The results show that the sneezing score was significantly lower for the Ap group than with the placebo group during the early period of pollen dispersion and during the main dispersion period. In addition, no adverse reactions were induced by Ap during the study. These results suggest that Ap may alleviate the symptoms of cedar pollinosis.
We examined the effects of di-D-fructose anhydride (DFA) III and IV on Ca absorption in luminally perfused segments of the small intestine in anesthetized rats. The calcium absorption rate with perfusion of 10 mmol/l CaCl2 was similarly increased by addition of 100 mmol/l DFAIII or IV, and these promotive effects of both DFAs were pronounced at perfusion rate of 0.15 ml/min than at 0.3 ml/min. The promotive effects were higher in the duodenojejunum than in the ileum.
A study was conducted to evaluate a net value of exogenous (dietary) protein nutrition in rats fed a [15N]-labeled soy protein isolate (SPI). Although [15N]-SPI-derived nitrogen reached a plateau 2 hr after feeding, it accounted for only a half of the total nitrogen in the small intestine. 15N was confirmed that was normally transported to liver, kidney, spleen, and brain. The present study reveals the large degree of participation of endogenous proteins in dietary SPI during small-intestinal digestion.
Bacillus brevis (Brevibacillus parabrevis) ATCC 8185 synthesizes two kinds of antibiotic peptides, cyclopeptide tyrocidine and linear gramicidin. The production of linear gramicidin can be induced by the standard method (using a skim milk medium for pre-culture and beef broth for the main culture) employed for the induction of tyrocidine. In this study, we tried to determine the optimal growth medium for B. brevis ATCC 8185 for synthesizing linear gramicidin. The yield of linear gramicidin produced by the standard method was 3.11 μg/ml. When beef broth was used both as the pre-medium and the main medium, the yield of the antibiotic was only 0.59 μg/ml. To confirm the influence of skim milk, the strain was grown in a 1% skim milk medium. As a result, the amount of linear gramicidin produced reached 20.3 μg/ml. These findings show the importance of skim milk in the production of linear gramicidin. In the skim milk medium, the cells produced an extracellular protease 2 h before the linear gramicidin was expressed. The 1% skim milk medium pretreated by this protease did not allow the induction of linear gramicidin into the cells, and protease activity was not detected in the supernatant of the culture. When the cells were cultivated in a 1% egg albumin medium, protease activity from the supernatant of the culture was detected, but production of linear gramicidin was not observed. Therefore, a 1% casein medium was used for production of linear gramicidin. As a result, the yield of linear gramicidin produced in the medium reached 6.69 μg/ml. We concluded that a digested product of the extracellular protease from casein enhances linear gramicidin production.
Growth of Escherichia coli K-12 in a modified Davis minimal medium was inhibited under high osmolarity, but it recovered remarkably with the addition of 1 mM proline. The co-existence of K+ with proline enhanced the recovery of growth under high osmolarity more than that in the presence of proline alone. The same was true for the activities of respiration and glucose uptake. A similar supplementary effect of K+ was observed for the activities of proline uptake under high osmolarity. These results suggest that K+ and proline support not only growth but respiration and uptake of the respiratory substrate glucose in the cell cytoplasm when exposed to high osmolarity. External K+ almost disappeared with 1 h of incubation at low osmolarity, indicating that active accumulation of K+ in the cells occurred. On the other hand, a gradual accumulation of K+ was recognized at high osmolarity in the presence of 1 M NaCl, especially at >2 h of incubation. This study of L-[5-3H]proline uptake in the cell cytoplasm indicates that proline was incorporated as a substrate of protein synthesis in the absence of NaCl, but was efficiently utilized as a compatible solute in the presence of high concentrations of NaCl.
Nuclear migration is indispensable for normal growth, differentiation, and development, and has been studied in several fungi including Aspergillus nidulans and Neurospora crassa. To better characterize nuclear movement and its consequences during conidiophore development, conidiation, and conidial germination, we performed confocal microscopy and time-lapse imaging on A. nidulans and Aspergillus oryzae strains expressing the histone H2B-EGFP fusion protein. Active trafficking of nuclei from a vesicle to a phialide and subsequently into a conidium provided the mechanistic basis for the formation of multinucleate conidia in A. oryzae. In particular, the first direct visual evidence on multinucleate conidium formation by the migration of nuclei from a phialide into the conidium, rather than by mitotic division in a newly formed conidium, was obtained. Interestingly, a statistical analysis on conidial germination revealed that conidia with more nuclei germinated earlier than those with fewer nuclei. Moreover, multinucleation of conidia conferred greater viability and resistance to UV-irradiation and freeze-thaw treatment.
Theanine was formed from glutamic acid and ethylamine by coupling the reaction of glutamine synthetase (GS) of Pseudomonas taetrolens Y-30 with sugar fermentation of baker’s yeast cells as an ATP-regeneration system. Theanine formation was stimulated by the addition of Mn2+ to the mixture for the coupling. The addition of Mg2+ was less effective. In a mixture containing a larger amount of yeast cells with a fixed level of GS, glucose (the energy source) was consumed rapidly, resulting in a decrease in the final yield of theanine. On the other hand, an increase in GS amounts increased theanine formation in a mixture with a fixed amount of yeast cells. High concentrations of ethylamine enhanced theanine formation whereas inhibited yeast fermentation of sugar and the two contrary effects of ethylamine caused a high yield of theanine based on glucose consumed. In an improved reaction mixture containing 200 mM sodium glutamate, 1,200 mM ethylamine, 300 mM glucose, 50 mM potassium phosphate buffer (pH 7.0), 5 mM MnCl2, 5 mM AMP, 100 units/ml GS, and 60 mg/ml yeast cells, approximately 170 mM theanine was formed in 48 h.
Chitinase B was purified from a culture medium of Ralstonia sp. A-471 by precipitation with (NH4)2SO4 and column chromatography with DEAE-Toyopearl 650M and Sephacryl S-200. The purified enzyme was homogeneous on SDS–PAGE. The molecular weight was 45,000 by SDS–PAGE. The optimum pH was 5.0 and stable pH was from 5.0 to 10.0. In the early stage of the reaction, chitinase B produced β-anomer of (GlcNAc)2 from the substrate (GlcNAc)6, whereas (GlcNAc)4 produced almost at equilibrium, indicating that the enzyme predominantly hydrolyzes the second glycosidic linkage from the nonreducing end of (GlcNAc)6.