Near-UV irradiation caused the decomposition of hinokitiol in an aqueous solution. During the photochemical reaction, the distinct electron spin resonance signal characteristic of the adduct of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) with the hydroxyl radical was accompanied by small signals corresponding to the adduct of DMPO with the superoxide anion radical. More than 95% of Escherichia coli cells were killed by the incubation with hinokitiol under near-UV irradiation by BLB fluorescent lamps. These results indicated the generation of reactive oxygen species during photochemical reaction of hinokitiol under near-UV irradiation.
The metabolism of deuterium-labeled (±)-jasmonicacid and 3-oxo-2-[(Z)pent-2′-enyl]-cyclopentan-1- octanoic acid in potato (Solanum tuberosum L.) was examined by using cultures of potato single-node stems. Deuterium-labeled (±)-jasmonic acid and 3-oxo-2-[(Z) pent-2′-enyl]cyclopentan-1-octanoic acid, which had been prepared from commercially available methyl (±)-jasmonate, were fed to the cultures, and the metabolites were extracted from the plants and analyzed by a liquid chromatography-selected ion monitoring system. The metabolism of deuterium-labeled (±)-jasmonic acid and 3-oxo-2-[(Z)-2′-pentenyl]cyclopentan-1-octanoic acid to 5′ and 4′-O-glucopyranosyloxyjasmoic acids was strongly suggested.
(R) and (S)-Aldehydes 2, which are intermediates for the synthesis of (5R) and (5S)-HETE, were respectively synthesized from the yeast-mediated reductive products, hydroxy ester 3 and cis-lactone 4, through Baeyer-Villiger oxidation with complete retention of enantiomeric excess.
The methanolic extract of Myrsine seguinii yielded the novel anti-inflammatory compound, myrsinoic acid E (1), whose structure was elucidated to be 3,5-digeranyl-4-hydroxy benzoic acid. We synthesized 1- and its 3,5-diprenyl (2) and 3,5-difarnesyl analogues (3). Compounds 1-3 suppressed 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation of mouse ears by 59%, 14%, and 69% at a dose of 1.4 μmol.
Ethyl 4-[2-(6-methyl-3-pyridyloxy)butyloxy]benzoate (2) was prepared as a novel anti-juvenile hormone (anti-JH) agent. Compound 2 induced precocious metamorphosis in larvae of the silkworm and black pigmentation of the larval cuticle, which are clearly recognized as JH-deficiency symptoms. The 4-ethoxycarbonyl group on the benzene ring was indispensable for activity. The activity of compound 2 could be fully counteracted by methoprene, a JH agonist, but not by the dietary administration of 20-hydroxyecdysone.
An extracellular 45 kDa endochitosanase was purified and characterized from the culture supernatant of Bacillus sp. P16. The purified enzyme showed an optimum pH of 5.5 and optimum temperature of 60°C, and was stable between pH 4.5-10.0 and under 50°C. The Km and Vmax were measured with a chitosan of a D.A. of 20.2% as 0.52 mg/ml and 7.71×10−6 mol/sec/mg protein, respectively. The enzyme did not degrade chitin, cellulose, or starch. The chitosanase digested partially N-acetylated chitosans, with maximum activity for 15-30% and lesser activity for 0-15% acetylated chitosan. The chitosanase rapidly reduced the viscosity of chitosan solutions at a very early stage of reaction, suggesting the endotype of cleavage in polymeric chitosan chains. The chitosanase hydrolyzed (GlcN)7 in an endo-splitting manner producing a mixture of (GlcN)2-5. Time course studies showed a decrease in the rate of substrate degradation from (GlcN)7 to (GlcN)6 to (GlcN)5, as indicated by the apparent first order rate constants, k1 values, of 4.98×10−4, 2.3×10−4, and 9.3×10−6 sec−1, respectively. The enzyme hardly catalyzed degradation of chitooligomers smaller than the pentamer.
Celastrol, which is a triterpene purified from Celastraceae plants, has anticancer and anti-inflammatory activities. In this study we investigated to clarify whether celastrol can induce apoptosis in a human leukemia HL-60 model system. Celastrol was found to induce apoptosis, and the rank order of the potency of celastrol and its derivatives to induce internucleosomal DNA fragmentation was found to be celastrol>cela-H>>the other derivatives=vehicle control. Many anticancer agents are known to possess the ability to inhibit topoisomerase II, so the inhibitory activities of celastrol and its derivatives on topoisomerase II were also explored. The rank order of the inhibitory activity was found to be celastrol>etoposide>cela-H, indicating that the apoptosis-inducing activities of cela derivatives correspond to their inhibitory activities on topoisomerase II. These data suggested that celastrol may cause its effects such as anticancer activity by the mechanism of apoptosis along with topoisomerase II inhibition.
Two kinds of isoforms of glucose 6-phosphate dehydrogenase (G6PDH) were purified from cells of a freezing-tolerant strain, Chlorella vulgaris C-27, by sequential steps of chromatography on five kinds of columns, including a HiTrap Blue column which showed excellent separation of the isoforms from each other. The two isoforms (G6PDH1 and G6PDH2) were purified up to 109-fold and 197-fold with specific activity of 14.4 and 26.0 U/mg-protein, respectively. G6PDH1 showed an apparent Mr of 200,000 with a subunit Mr of about 58,000, whereas G6PDH2 showed an apparent Mr of 450,000 with a subunit Mr of about 52,000. The kinetic parameters were measured and several enzymatic features of the isoforms, such as effects of metal ions on the enzyme activity, were clarified, which showed that the two isoforms were different from each other in many respects. Among the effective ions, Cd2+ showed marked stimulating effects on both isoforms. G6PDH1 and G6PDH2 seem to be a cytosolic and a chloroplastic type, respectively, as judged by their sensitivity to DTT, and also from the results of sequence similarity searches using their N-terminal and internal amino acid sequences.
Theonellapeptolide Ie (Tp), an oligopeptide lactone isolated from a marine sponge, Petrosia sp., was shown to induce an unprecedented morphological change in the immature oocytes of the starfish Asterina pectinifera. The cortical F-actin was disturbed and assembled to form dots and rings, as evidenced by staining with rhodamine-conjugated phalloidin. The oocyte eventually became malformed. When Tp was added to an immature oocyte which had been pretreated with cytochalasin B or D, inhibitors of actin polymerization, no malformation was observed. When Tp was added to an oocyte which had been induced to mature by 1-methyladenine (1-MeAde), a maturation-inducing substance in starfishes, no morphological changes were observed in the maturing oocytes which reached the first meiotic prometaphase 40 min after the start of 1-MeAde treatment. This is the first description of a chemical that induces aberrant redistribution of F-actin-based cytoskeleton in an animal oocyte which is arrested at the first meiotic prophase.
A series of benz[f]indole-4,9-diones, based on the antitumor activity of 1,4-naphthoquinone, were synthesized and evaluated for their cytotoxic activity in cultured human cancer cell lines A549 (lung cancer), Col2 (colon cancer), and SNU-638 (stomach cancer), and also for the inhibition of human DNA topoisomerases I and II activity in vitro. Several compounds including 2-amino-3-ethoxycarbonyl-N-methyl-benz[f]indole-4,9-dione showed a potential cytotoxic activity judged by IC50<20.0 μg/ml in the panel of cancer cell lines. Especially, 2-hydroxy-3-ethoxycarbonyl-N-(3,4-dimethylphenyl)-benz[f]indole-4,9-dione had potential selective cytotoxicity against lung cancer cells (IC50=0.4 μg/ml)) compared to colon (IC50>20.0 μg/ml) and stomach (IC50>20.0 μg/ml) cancer cells. To further investigate the cytotoxic mechanism, the effects of test compounds on DNA topoisomerase I and II activities were used. In a topoisomerase I-mediated relaxation assay using human placenta DNA topoisomerase I and supercoiled pHOTI plasmid DNA, 2-amino-3-ethoxycarbonyl-N-(4-fluorophenyl)-benz[f]indole-4,9-dione had the most potent inhibitory activity among the compounds tested. However, most of the compounds showed only weak inhibition of the DNA topoisomerase II-mediated KDNA (Kinetoplast DNA) decatenation assay, except for 2-amino-3-ethoxycarbonyl-N-(4-methylphenyl)-benz[f]indole-4,9-dione and 2-amino-3-ethoxycarbonyl-N-(2-bromoehtyl)-benz[f]indole-4,9-dione with a moderate inhibitory activity. These results suggest that several active compounds had relatively selective inhibitory activity against toposiomearse I compared to toposiomerase II. No obvious correlation was observed between the cytotoxicity of the individual compound and the inhibitory activity of DNA relaxation and decatenation by topoisomerase I and II, respectively, in vitro.
NtrC protein of piezophilic Shewanella violacea was overexpressed and purified, to confirm the protein-DNA interaction. An electrophoretic mobility shift assay demonstrated that the NtrC recognizes the sequence for NtrC binding within the region upstream of the glnA operon. Western blot analysis also showed that the NtrC is expressed at a higher level under high-pressure conditions than under atmospheric pressure conditions.
A fluorescence quenching study of a sole tryptophan residue of a bifunctional alginate lyase from Pseudoalteromonas sp. strain No. 272 was done in the presence and absence of substrates, oligomeric guluronic and its C5 isomer mannuronic acid, by a Stern-Volmer plot with a quencher, acrylamide. N-Acetyltryptophanamide and reduced and carboxymethylated alginate lyase showed large quenching constants, on the other hand, the native enzyme had small constants regardless of the presence or absence of the substrates. The result suggests that the tryptophan residue is located in a buried region of the enzyme molecule, but is barely accessible to acrylamide, and that the residue is not masked by the substrates with various degrees of polymerization.
We estimated the subsite structure of the maltogenic amylase from Thermomonospora viridis TF-35 (TVA). TVA has six subsites, and the catalytic site is between the 3rd and 4th subsite. The subsite affinities, A-3, A-2, (A-1+A+1), A+2 and A+3 were calculated to be 0.47, 2.31, 0.49, 2.45, and 0.17 kcal mol-1, respectively.
Allosamidin is a strong inhibitor of family 18 chitinases. We previously reported the presence of allosamidin-sensitive and -insensitive chitinases (chitinase S and IS) in the culture filtrate of the allosamidin-producing strain, Streptomyces sp. AJ9463. In this study, we cloned and sequenced the genes encoding the two chitinases, which clarified that chitinase S and IS belong to the family 18 and 19 chitinase, respectively.
We cloned a gene for the iron sulfur protein (Ip) subunit from an edible mushroom, Lentinula edodes, and introduced a point mutation that confers carboxin resistance into it. The mutant gene successfully transformed L. edodes with high efficiency (9 transformants/2.5 μg vector DNA). Restriction enzyme-mediated integration (REMI) increased the transformation efficiency by about two-fold.
The root elongation activity of unsaturated oligomeric uronates from alginate on carrot and rice plants was investigated. Unsaturated oligomeric uronates were prepared by digesting polymannuronate (PM) and polyguluronate (PG) with an alginate lyase purified from Pseudoalteromonas sp. strain No. 272. The root elongation activity was measured by elongation in length of carrot- and rice-excised root incubated in the B5-medium containing 0.8% agar in the dark. PM and PG showed no activity, but the enzymatic digestion mixtures of PG had promoting activity on roots of both plants at a final concentration of 0.5 mg/ml. The maximum activity was obtained at 0.75 mg/ml. The dependence of activity on degree of polymerization of the uronates was tested and the pentamer was most active, but the mechanism of the action of unsaturated uronates on the cells remains to be solved.
We introduce the TA cloning antibody method for the high-fidelity PCR product amplified by family B DNA polymerase without purification. This method uses antibodies and Thermus aquaticus (Taq) DNA polymerase. The antibodies can inhibit only the activity of family B DNA polymerase, and Taq can co-work for A-tailing. This method has nearly cloning efficiency to that of the PCR product of Taq.
Western blotting coupled with immunoprecipitation showed that activin A treatment induced phosphorylation of Smad2 but not complex formation of Smad2/4 in human colon cancer-derived HT-29 cells. Because HT-29 cells expressed neither Smad4 mRNA nor Smad4 protein, it is suggested that deletion of Smad4 leads to a defect of formation of Smad2/4 complex upon activin A stimulation in HT-29 cells.
The stepwise chromatographic behaviour on DEAE-Sepharose of rat Fe65, a neuronal protein, was tested, using as eluants KCl, CaCl2, and MgCl2. Assays by western blot showed that Fe65 was eluted by CaCl2, at a ionic strength 20% lower than that of MgCl2 or KCl. Interestingly, in the case of a truncated Fe65, lacking a glutamic acid rich region at the N-terminus, the ionic strengths of the various eluants were almost identical. These results suggested a possible inhibitory role of calcium ions in the binding of the protein to DEAE and a specific affinity of these ions for long acidic stretches.
To develop a convenient method to isolate lectins, we prepared an affinity gel by coupling plasma proteins with agarose beads under conditions where the pH did not exceed 7.5. The validity of the use of this affinity gel in combination with elution using a hapten saccharide was confirmed by isolation of concanavalin A from Jack bean meal. Successful application of the method was demonstrated by isolation of two novel vegetable lectins from udo (Aralia cordate) and wasabi (Wasabia japonica). The method would be useful to isolate new lectins from various sources including plant and animal tissues.
While doing a structural analysis of minor component N-glycans linked to 350-kDa royal jelly glycoprotein (RJGP), which stimulates the proliferation of human monocytes, we found that a Galβ1-3GlcNAcβ1-4Man unit occurs on the insect glycoprotein. The structure of the fluorescence-labeled N-glycan was analyzed by sugar component analysis, IS-MS, and 1H-NMR. The structural analysis showed that the 350-kDa RJGP bears Galβ1-3GlcNAcβ1-4(GlcNAcβ1-2)Manα1-3 (Manα1-3Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAc, suggesting this insect glycoprotein is one of the substrates for both β1-3 galactosyl and β1-4 N-acetylglucosamininyl transferases. To our knowledge, this is the first report that succeeded in identifing an insect glycoprotein bearing the β1-3 galactosylated N-glycan.
In female SD rats that were injected with 4 g/kg BW ethanol p.o. followed by a 5 mg/kg BW lipopolysaccharide (LPS) i.v. injection, serum glutamic pyruvic transaminases (GPT) activity increased to about eight times that of normal rats. In this model, rats that had been fed a diet containing 1% Hydrangeae Dulcis Folium (HDF) extracts for fifteen days showed significantly lower serum GPT activity (380.0±58.2 IU/l) than the control group (3527.0±774.1 IU/l). HDF's efficacy was far superior to milk thistle in this model (2950.0±915.9 IU/l). When mouse macrophages were treated with HDF extracts at 50 μg/ml, TNF-α production induced by LPS was suppressed to about 10% of the control. Rat serum TNF-α levels induced by LPS was decreased to 58.7% of the control by administering 1000 mg/kg BW HDF extract p.o. These results indicate that HDF prevents alcohol-induced liver injury through the inhibition of TNF-α production.
Soluble soybean polysaccharide (SSPS) was fractionated into its low- (LMW) and high-molecular-weight (HMW) components to test their antioxidative and emulsifying properties. Linoleic acid was emulsified with an aqueous solution of SSPS, HMW, a mixture of LMW or HMW with maltodextrin, or maltodextrin alone. The emulsions prepared with SSPS, HWM and the mixture of HMW with maltodextrin were stable. These emulsions were spay-dried to produce microcapsules. The encapsulated linoleic acid was oxidized at 37°C and at various levels of relative humidity. Linoleic acid encapsulated with the mixture of LMW with maltodextrin or HMW was stable to oxidation, and this stability increased as the weight fraction of LMW in the mixture was increased. The LMW components also had high DPPH-radical scavenging activity. These results indicate that LMW played an important role in suppressing or retarding the oxidation of linoleic acid encapsulated with SSPS. The oxidative stability of linoleic acid encapsulated with a mixture of the LMW and HMW components was high at low and high relative humidity, but not at intermediate levels of relative humidity.
The oxindole-3-acetic acids, oxidative metabolites of indole-3-acetic acid, were isolated from a byproduct of a corn starch manufacturing plant, and were further converted to the 3-hydroxyl derivatives in the presence of metal ion. The mechanical study was followed by a chemical analysis including other byproducts, and suggested the presence of an intermediate that had a radical at the C-3 position of oxindole-3-acetic acids.
The chicken egg white ovoinhibitor, a multi-type proteinase inhibitor, was conjugated with galactomannan through the Maillard reaction in a controlled dry heating state at 60°C and 65% relative humidity. The formation of an ovoinhibitor-galactomannan conjugate during dry heating was confirmed by SDS-PAGE. The resulting ovoinhibitor-galactomannan conjugate showed almost the same inhibitory activity toward trypsin, chymotrypsin and elastase as that of the untreated ovoinhibitor, while the conjugate showed stronger heat stability and better emulsifying properties than the untreated ovoinhibitor. These results suggest that the ovoinhibitor-galactomannan conjugate can be used as a protease inhibitor having heat stability and outstanding emulsifying properties for industrial application.
This study was designed to investigate whether genistein may ameliorate oxidative stress and nuclear factor κB (NFκB) activation in the lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophage cell line. Treatment of RAW 264.7 cells with genistein significantly reduced lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production in a dose-dependent manner with an IC50 of 69.4 μM. Genistein at 50 μM and 100 μM concentrations reduced thiobarbituric acid-reactive substances (TBARS) accumulation, increasing the GSH level and antioxidant enzyme activities, such as superoxide dismutase (SOD) and catalase. The specific DNA-binding activities of nuclear factor κB (NFκB) on nuclear extracts from 50 μM and 100 μM genistein treatments were significanly suppressed. These results suggest that genistein has mild antioxidant activity to suppress intracellular oxidative stress and NFκB activation.
Noise is one of the factors that induces critical stress in animals. The contents of glycogen, lactic acid and cholesterol in the liver of noise-stressed rats were analyzed in order to investigate the alleviation of noise-stress-induced physiological damages by traditional medicine using Astragali and Rhodiolae radices. More than 95 dB noise ranging from 2 to 4 kHz reduced the contents of these compounds in the liver of rats not injected with the extract of Astragali or Rhodiolae, but did not change the contents in the liver of rats injected with the Astragali or Rhodiolae extract. These results show that noise induced stress in the rats via a decrease in contents of these compounds in the liver and that Astragali or Rhodiolae maintained the contents of these compounds in the liver of the noise-stressed rats. The results indicate that Astragali or Rhodiolae improved the ability for rats to resist noise stress.
The purpose of this study is to examine the effects of highly purified structured lipids on serum and liver lipid profiles in rats. We also investigated in vitro hydrolysis of lipid emulsions by porcine pancreas. Hydrolysis rates of medium chain (M)-linoleic (L)-medium chain (M) types were 2 to 3 times higher than those of L-M-L types. The diet containing structured lipids or corn oil was administered to rats for 4 weeks. There were no significant differences in growth and food efficiency. Serum cholesterol levels were significantly lower (P<0.05) in the 2-octanoyl-1,3-dilinoleoyl-glycerol, 2-linoleoyl-1,3-didecanoyl-glycerol, and 2-decanoyl-1,3-dilinoleoyl-glycerol groups than in the corn-oil group. Serum triglyceride levels were significantly lower (P<0.05) in rats fed L-M-L types than those in the other groups. Serum non-esterified fatty acid (NEFA) and β-hydroxybutylate levels were significantly higher (P<0.01) in rats fed M-L-M types than those of the other groups. These results indicate that the feeding of highly purified L-M-L types could effectively improve serum and liver lipid profiles and that M-L-M types may be a preferable substrate for the pancreas and contribute to energy supply in rats.
The brown alga, Cladosiphon okamuranus TOKIDA, was found to contain a large amount of free fatty acid (45% of the total lipids). A crude enzyme preparation from the alga showed activity for hydrolyzing the acyl groups of various glycerolipids. The results suggest that the free fatty acid in C. okamuranus was released mainly from glycoglycerolipids, which were the major lipid components in the alga, by such glycerolipid acyl hydrolases as galactolipase.
We examined the effects of (−)-Hydroxycitrate (HCA) ingestion on fat oxidation during moderate intensity exercise in untrained men. Six subjects ingested 500 mg of HCA or a placebo for 5 days and did endurance exercise. Blood FFA concentrations were significantly increased and respiratory exchange ratio (RER) decreased by HCA ingestion. These results suggested short-term HCA ingestion increases fat oxidation in untrained men.
The structural specificity of the monocarboxylic acid transporter (MCT) for the transport of phenolic acids was investigated by measuring the inhibitory effect on the fluorescein transport in Caco-2 cell monolayers. Although most of the monohydroxylated derivatives had an inhibitory effect, the di- and tri-hydroxylated ones did not. The methoxylated derivatives were more inhibitory than the hydroxylated ones in all the meta-substituted derivatives, suggesting that meta-hydroxylation of the substrate would decrease the affinity for MCT.
Two proteases capable of decreasing dough strength when added to wheat flour were purified from Maitake and these were both thought to be peptidyl-Lys metalloendopeptidase. The major purified protease SP-3-A hydrolyzed high-molecular-weight glutenin subunits preferably to the other glutenin subunits. SP-3-A cleaved peptide bonds adjacent to the N-terminal of lysine in the high-molecular-weight glutenin subunit.
A potent producer of D-arabitol was isolated by screening of natural sources and identified as Metschnikowia reukaufii AJ14787. Resting cells of this strain can efficiently produce D-arabitol from D-glucose with a weight yield of more than 60%, and can also produce D-arabitol from several other types of sugars such as polyols, ketoses, and aldoses. To improve productivity, various culture conditions such as temperature and the concentrations of D-glucose and nitrogen sources were examined. Under optimal conditions, 206 g/l of D-arabitol was produced from D-glucose with a weight yield of 52% in 100 hours.
Some ice-nucleating bacterial strains, including Pantoea ananatis (Erwinia uredovora), Pseudomonas fluorescens, and Pseudomonas syringae isolates, were examined for the ability to shed ice nuclei into the growth medium. A novel ice-nucleating bacterium, Pseudomonas antarctica IN-74, was isolated from Ross Island, Antarctica. Cell-free ice nuclei from P. antarctica IN-74 were different from the conventional cell-free ice nuclei and showed a unique characterization. Cell-free ice nuclei were purified by centrifugation, filtration (0.45 μm), ultrafiltration, and gel filtration. In an ice-nucleating medium in 1 liter of cell culture, maximum growth was obtained with the production of 1.9 mg of cell-free ice nuclei. Ice nucleation activity in these cell-free ice nuclei preparations was extremely sensitive to pH. It was demonstrated that the components of cell-free ice nuclei were protein (33%), saccharide (12%), and lipid (55%), indicating that cell-free ice nuclei were lipoglycoproteins. Also, carbohydrate and lipid stains showed that cell-free ice nuclei contained both carbohydrate and lipid moieties.
The bph operon of Pseudomonas sp. KKS102 is constituted of 11 bph genes which encode enzymes for biphenyl assimilation. Growth of a mutant in which a large part of the bph operon was deleted was inhibited by biphenyl in a concentration-dependent manner. We constructed a series of bph operon deletion mutants and tested for their biphenyl sensitivity. Growth inhibition by biphenyl was more prominent with the mutants defective in bphA1, bphB, bphC, and bphD, which were clustered in the bph operon and working in the early stage of the biphenyl degradation. The mutant defective in bphE, which was working at the late stage and forming a different cluster from the early stage genes, was not much inhibited by biphenyl. These indicate that biphenyl is detoxified by enzymes which function in the early stage of biphenyl assimilation and thus detoxification of substrates as well as energy acquisition could have played an important role in the evolution of the KKS102 bph operon.
KA-prep, a culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation (CWP) of Schizophyllum commune, has been reported to have an activity to form protoplasts from S. commune mycelia. The SDS-polyacrylamide gel electrophoreses described here demonstrated that a specific proteinous component (molecular weight: 150,000) occurred in KA-prep. The protein (P150T) was also formed in culture filtrates with CWP of several basidiomycetes, which could release the protoplasts, suggesting that the component was an indispensable factor for protoplast formation. P150T, isolated from an ammonium sulfate fraction of KA-prep (0-30% saturation), did not have any protoplast-forming activity. Results were obtained indicating that P150T participates in protoplast formation together with chitinase(s) and β-glucanase(s) in KA-prep. The N-terminal amino acid sequence indicated an analogy of P150T to mutanase (α-1,3-glucanase) from Bacillus sp. RM1, and actually P150T hydrolyzed mutan as well as S-(α-1,3) glucan from S. commune.
Three endoglucanase genes (cel5A, cel5B, and cel61A) were cloned from an industrial fungus, Aspergillus kawachii. Yeasts transformed with these cDNAs showed endoglucanase activity in medium. Cel5A and Cel61A contained a type 1 cellulose-binding domain (CBD1) at the C-terminus of the enzyme. The putative catalytic regions of Cel5A and Cel5B showed homology with various endoglucanases belonging glycosyl hydrolase family 5 (GH5). Cel5B showed high homology with Cel5A in catalytic region, but it lacked CBD1 and linker. The cel5A contained four introns, whereas cel5B contained five introns. The putative catalytic region of Cel61A showed homology with enzymes belonging to GH61. The cel61A contained no introns.
New phenol degrading bacteria with high biodegradation activity and high tolerance were isolated as Burkholderia cepacia PW3 and Pseudomonas aeruginosa AT2. Both isolates could grow aerobically on phenol as a sole carbon source even at 3 g/l. The whole-cell kinetic properties for phenol degradation by strains PW3 and AT2 showed a Vmax of 0.321 and 0.253 mg/l/min/(mg protein), respectively. The metabolic pathways for phenol biodegradation in both strains were assigned to the meta-cleavage activity of catechol 2,3-dioxygenase.
Adenylate cyclase deficient (cya) mutants of E. coli K-12 were found to be resistant to fosmidomycin, a specific inhibitor of the non-mevalonate pathway, just like to fosfomycin. E. coli glpT mutants were resistant to fosfomycin and also to fosmidomycin. This fact shows that fosmidomycin was transported inside via the glycerol-3-phosphate transporter, GlpT. DNA micro-array analysis showed that the transcription of glpT and other genes concerning glycerol utilization were highly dependent on the presence of cAMP.