(1S,4R,5R)-(+)-α-Acoradiene [1,8-dimethyl-4-(1-methylethenyl)spiro[4.5]dec-7-ene] (1) is shown by its synthesis to be the major component of the male-produced aggregation pheromone of the broad-horned flour beetle (Gnatocerus cornutus).
(+) and (−)-Dihydropinidine and (+)- and (−)-epidihydropinidine were synthesized from hydroxy esters 1 and 2 which had been prepared by yeast reduction of methyl (2-oxocyclohexyl)acetate. The enantiomeric excess at the C-1 positions of 1 and 2 were both determined as more than 99% ee.
To provide insight into the catalytic mechanism for the final deprotonation reaction of squalene-hopene cyclase (SHC) from Alicyclobacillus acidocaldarius, mutagenesis experiments were conducted for the following ten residues: Thr41, Glu45, Glu93, Arg127, Trp133, Gln262, Pro263, Tyr267, Phe434 and Phe437. An X-ray analysis of SHC has revealed that two types of water molecules (“front water” and “back waters”) were involved around the deprotonation site. The results of these mutagenesis experiments allow us to propose the functions of these residues. The two residues of Gln262 and Pro263 probably work to keep away the isopropyl group of the hopanyl cation intermediate from the “front water molecule,” that is, to place the “front water” in a favorable position, leading to the minimal production of by-products, i.e., hopanol and hop-21(22)-ene. The five residues of Thr41, Glu45, Glu93, Arg127 and Trp133, by which the hydrogen-bonded network incorporating the “back waters” is constructed, increase the polarization of the “front water” to facilitate proton elimination from the isopropyl moiety of the hopanyl cation, leading to the normal product, hop-22(29)-ene. The three aromatic residues of Tyr267, Phe434 and Phe437 are likely to play an important role in guiding squalene from the enzyme surface to the reaction cavity (substrate channeling) by the strong affinity of their aromatic residues to the squalene substrate.
Three new communesin congeners, communesins C, D, and E, together with two known communesins A and B were isolated from okara that had been fermented with Penicillium expansum Link MK-57. All these communesins showed the insecticidal activity against the silkworms.
To compare the actions of clothianidin, a neonicotinoid acting on insect nicotinic acetylcholine receptors, and related compounds with that of imidacloprid, the compounds were tested on the Drosophila SAD-chicken β2 nicotinic acetylcholine receptor expressed in Xenopus laevis oocytes using two-electrode voltage-clamp electrophysiology. The maximum response of the SADβ2 nicotinic receptor to clothianidin was larger than that observed for acetylcholine. Ring breakage of the imidazolidine ring of imidacloprid resulting in the generation of a guanidine group was critical for this super agonist action.
The reactivity of N-tosylindole (4) in the presence of aluminum chloride was studied, and two types of oligomerization of 4 were observed. One type was condensation between both pyrrole parts (dimers 5 and 6 and trimer 7) and the other was between a pyrrole part and a benzene part of each indole nucleus (dimers 8 and 9).
We examined the role of SigC (Sll0184), a sigma factor of RNA polymerase (RNAP), in a unicellular cyanobacterium, Synechocystis sp. strain PCC 6803. On the inactivation of sigC, which is an Escherichia coli rpoD homolog, cells were viable but had a low survival rate in the stationary phase of growth under normal physiological conditions, indicating that SigC is a group 2 type sigma factor. In analyses of transcript and protein levels using the sigC knockout strain, it was found that expression of glnB, a nitrogen key regulatory gene, is controlled by SigC in the stationary phase. Primer extension revealed that the glnB nitrogen promoter (P2) was specifically recognized by SigC in the stationary phase under conditions of nitrogen starvation. In vitro studies with purified enzymes indicated effective transcription, on supercoiled DNA templates, from P2 by SigC-RNAP with NtcA which is an activator for nitrogen gene transcription. DNase I footprinting also indicated binding and related sites of NtcA and/or RNAP with SigC on the nitrogen promoter. The unique promoter architecture and the mechanism of transcription by RNAP with SigC are also discussed.
The 70-kDa protoxin of Cry11A, a dipteran-specific insecticidal protein, was processed by trypsin into 36- and 32-kDa fragments. To investigate the potent function of the two processed fragments, a GST (Glutathione-S-transferase) fusion protein of each polypeptide was constructed. While neither the 36- nor the 32-kDa fragment was toxic to Culex pipiens larvae, coexpression of the two fragments restored the insecticidal activity. Furthermore, the coprecipitation experiment demonstrated that the 36-kDa fragment was associated with the 32-kDa fragment. It was, therefore, shown that the coexistence of the two processed fragments of Cry11A was essential for the toxicity. The mutant of the 36-kDa fragment lacking the region from Gly257 to Arg360 bound to the 32-kDa fragment but the coexpression with the 32-kDa fragment resulted in no toxicity, suggesting that this region was involved in insecticidal activity.
A secreted luciferase from the marine ostracod, Vargula hilgendorfii, is a useful tool for gene expression assays in living mammalian cells. We have cloned the cDNA of a new secreted luciferase from the ostracod Cypridina noctiluca, which inhabits the coast of Japan. C. noctiluca luciferase consists of 553 amino acid residues with a molecular mass of 61,415 Da, as deduced from the nucleotide sequence. The homologies of nucleotide and amino acid sequences with V. hilgendorfii luciferase are 79.2% and 83.1%, respectively. C. noctiluca luciferase can expressed in and secreted from cultured mammalian cells. The characteristic properties of expressed C. noctiluca luciferase are similar to those of V. hilgendorfii luciferase. However, the activity of C. noctiluca luciferase in culture medium is much higher than that of V. hilgendorfii luciferase, suggesting that C. noctiluca luciferase is a highly potent reporter enzyme for real-time and continuous monitoring of gene expression in living cells.
Novel antimicrobial peptides (AMP), designated Tu-AMP 1 and Tu-AMP 2, were purified from the bulbs of tulip (Tulipa gesneriana L.) by chitin affinity chromatography and reverse-phase high-performance liquid chromatography (HPLC). They bind to chitin in a reversible way. They were basic peptides having isoelectric points of over 12. Tu-AMP 1 and Tu-AMP 2 had molecular masses of 4,988 Da and 5,006 Da on MALDI-TOF MS analysis, and their extinction coefficients of 1% aqueous solutions at 280 nm were 3.3 and 3.4, respectively. Half of all amino acid residues of Tu-AMP 1 and Tu-AMP 2 were occupied by cysteine, arginine, lysine, and proline. The concentrations of peptides required for 50% inhibition (IC50) of the growth of plant pathogenic bacteria and fungi were 2 to 20 μg/ml. The structural characteristics of Tu-AMP 1 and Tu-AMP 2 indicated that they were novel thionin-like antimicrobial peptides, though Tu-AMP 2 was a heterodimer composes of two short peptides joined with disulfide bonds.
To investigate the functional role of subsites E and F in lysozyme catalysis, Asn37 of hen egg-white lysozyme (HEL), which is postulated to participate in sugar residue binding at the right-sided subsite F through hydrogen bonding, was replaced by Ser or Gly by site-directed mutagenesis. The mutations of Asn37 neither significantly affected the binding constant for chitotriose nor the enzymatic activity toward the substrate glycol chitin. However, kinetic analysis with the substrate N-acetylglucosamine pentamer, (GlcNAc)5, revealed that the conversion of Asn37 to Gly decreased the binding free energies for subsites E and F, while the conversion to Ser increased the substrate affinity at subsite F. It was further found that the rate constant of transglycosylation was reduced by these mutations. These results suggest that Asn37 is involved not only in substrate binding at subsite F but also in transglycosylation activity. No remarkable change in the tertiary structure except the side chain of the 37th residue was detected on X-ray analysis of the mutant proteins, indicating that the alterations in the enzymatic function between the wild type and mutant enzymes depend on limited structural change around the substitution site. It is thus speculated that the slight conformational difference in the side chain of position 37 may affect the substrate and acceptor binding at subsites E and F, leading to lower the efficiency of the transglycosylation activities of the mutant proteins.
In Arabidopsis thaliana (L.) Heynh ecotype Landsberg, levels of soluble acid invertase activity are closely related to the progress of seed germination. To study the mechanism(s) of the development of these enzymes, two cDNA clones that encode putative vacuolar acid invertases were isolated from germinating seeds and very young seedlings using reverse-transcription polymerase chain reactions with degenerate primers. These fragments corresponded to the genes Atβfruct3 and Atβfruct4 from the Columbia ecotype. An apoplasmic invertase gene corresponding to Atβfruct1/ATCWINV1 was also isolated from these samples. Northern blot analyses showed that Atβfruct3 and Atβfruc4 are expressed concomitantly with germination and the subsequent seedling growth. In contrast, the Atβfruct1/AtcwINV1 mRNA is translated before germination. These expression patterns are regulated by phytochrome, which perceives red light and in turn triggers de novo synthesis of gibberellin, initiating Arabidopsis seed germination. To test the effects of gibberellin on the expression of these genes, seed were treated with a gibberellin biosynthesis inhibitor, uniconazole or prohexadione. These chemicals inhibited both seed germination and expression of the above genes, but subsequently applied GA4, an active gibberellin, reversed the inhibition. These results suggest that the transcription of genes encoding the vacuolar invertases, Atβfruct3 and Atβfruct4 and a gene encoding the apoplasmic invertase, Atβfruct1/AtcwINV1, are induced by gibberellin synthesized de novo following irradiation with red light.
Carpropamid ((1RS,3SR)-2,2-dichloro-N-[(R)-1-(4-chlorophenyl)ethyl]-1-ethyl-3-methylcyclopropanecarboxamide) is a potent chemical against the rice blast fungus, Pyricularia oryzae. In 2001, isolates of the fungus with reduced sensitivity to this fungicide appeared in Saga Prefecture of Japan and were regarded as a potential threat to rice protection by carpropamid. The cause of the resistance has been identified genetically as a point mutation resulting in the Val75Met change in scytalone dehydratase, the primary target of the fungicide. We constructed an overexpression system of the variant enzyme and characterized the kinetics in the catalysis and the inhibition by carpropamid isomers. The variant enzyme retained a significant level of enzymatic activity. Inhibition of the variant enzyme by carpropamid was more than 200-fold reduced in comparison with that of the wild-type. Based on the results, here we propose possible mechanisms of the carpropamid-resistance of the variant enzyme in retaining the normal enzymatic activity.
Two full-length cDNAs encoding gibberellin 3-oxidases, InGA3ox1 and InGA3ox2, were cloned from developing seeds of morning glory (Ipomoea nil (Pharbitis nil) Choisy cv. Violet) with degenerate-PCR and RACEs. The RNA-blot analysis for these clones revealed that the InGA3ox2 gene was organ-specifically expressed in the developing seeds at 6–18 days after anthesis. In situ hybridization showed the signals of InGA3ox2 mRNA in the seed coat, suggesting that active gibberellins (GAs) were synthesized in the tissue, although no active GA was detected there by immunohistochemistry. In situ hybridization analysis for InAmy1 (former PnAmy1) mRNA showed that InAmy1 was also synthesized in the seed coat. Both InGA3ox2 and InAmy1 genes were expressed spatially overlapped without a clear time lag, suggesting that both active GAs and InAmy1 were synthesized almost simultaneously in seed coat and secreted to the integument. These observations support the idea that GAs play an important role in seed development by inducing α-amylase.
In order to reduce the protein content of wastewater, photosynthetic bacteria producing proteinases were screened from wastewater of various sources and stocked in culture. An isolated strain, KDDS1, was identified as Rubrivivax gelatinosus, a purple nonsulfur bacterium that secretes proteinase under micro-aerobic conditions under light at 35°C. Molecular weight of the purified enzyme was estimated to be 32.5 kDa. The enzyme showed the highest activity at 45°C and pH 9.6, and the activity was completely inhibited by phenylmethyl sulfonyl fluoride (PMSF), but not by EDTA. The amino-terminal 24 amino acid sequence of the enzyme showed about 50% identity to those of serine proteinases from Pseudoalteromonas piscicida strain O-7 and Burkholderia pseudomallei. Thus, the enzyme from Rvi. gelatinosus KDDS1 was thought to be a serine-type proteinase. This was the first serine proteinase characterized from photosynthetic bacteria.
K5-type yeast killer protein in the culture supernatant of Pichia anomala NCYC 434 cells was concentrated by ultrafiltration and purified to homogenity by ion-exchange chromatography with a POROS HQ/M column followed by gel filtration with a TSK G2000SW column. The protein migrated as a single band on discontinous gradient SDS-PAGE and had a molecular mass of 49 000 Da. The pI value of the K5-type killer protein was measured at pH 3.7 by high voltage vertical gel electrofocusing. The result of an enzyme immuno assay revealed that it was a glycosylated protein. Its internal amino acid sequencing yielded the sequences LNDFWQQGYHNL, IPIGYWAFQLLDNDPY, and YGGSDYGDVVIGIELL, which are 100% identical to exo-β-1,3-glucanase (accession no. AJ222862) of Pichia anomala (strain K). The purified protein was highly stable at pH values between 3 and 5.5 and temperatures up to 37°C.
The plastid genome of higher plants contains more than one hundred genes for photosynthesis, gene expression, and other processes. Plastid transcription is done by two types of RNA polymerase, PEP and NEP. PEP is a eubacteria-type RNA polymerase that is essential for chloroplast development. In Arabidopsis thaliana, six sigma factors (SIG1-6) are encoded by the nuclear genome, and postulated to determine the transcription specificity of PEP. In this study, we constructed a DNA microarray for all of the plastid protein-coding genes, and analyzed the effects of the sig2 lesion on the global plastid gene expression. Of the 79 plastid protein genes, it was found that only the psaJ transcript was decreased in the mutant, whereas transcripts of 47 genes were rather increased. Since many of the up-regulated genes are under the control of NEP, it was suggested that the NEP activity was increased in the sig2-1 mutant.
Three bands of proteinase activity (Rf values of 0.5, 0.6, and 0.7) were detected on activity-stained gels after native gel electrophoresis of carrot (Daucus carota L. cv US-Harumakigosun) suspension cells. After the induction of somatic embryogenesis, one activity band (0.7 band) rapidly disappeared; the 0.6 band was absent at the heart-shaped embryo stage. However, the intensity of the 0.5 band increased during embryogenesis. An additional band (0.25 band) appeared after the torpedo-shaped stage. Three bands (0.25, 0.5, and 0.6) were also detected in zygotic seeds. Two activity bands (0.5 and 0.6) were classified as cysteine proteinases based on sensitivities to N-Ethylmaleimide (NEM) or L-3-trans-Carboxyoxirane-2-Carbonyl-L-Leucyl-Agmatine (E-64). To find candidate genes for the cysteine proteinases, we cloned seven cDNAs encoding putative cysteine proteinases from suspension cells and developing somatic embryos. The expression patterns of the seven genes were categorized into three types (Type A, mRNAs increase concomitantly with somatic embryogenesis; Type B, mRNAs decrease quickly in organized cells; Type C, no significant change in transcript level during somatic embryogenesis).
In an effort to elucidate the molecular mechanisms of fatty acid desaturation in yeast, a complete gene encoding Δ12 fatty acid desaturase of Saccharomyces kluyveri was cloned. The open reading frame of this gene (named Sk-FAD2) consists of 1,251 bp, encoding 416 amino acids. The deduced Sk-FAD2 protein had 37–55% identity with those from other filamentous fungi. Unlike the genes of these other fungi, S. cerevisiae expressing Sk-FAD2 was found to be capable of synthesizing the dienoic fatty acid hexadecadienoic acid as well as linoleic acid. Moreover, the Sk-FAD2-disrupted strain of S. kluyveri was unable to produce polyunsaturated fatty acids. These results suggested that Sk-FAD2 protein is a unique Δ12 fatty acid desaturase in S. kluyveri. Analysis of transcriptional expression revealed that Sk-FAD2 was not repressed by exogenous unsaturated fatty acids but responded to low-temperature stress.
We describe the genetic analysis of the bio operon of the biotin auxotrophic Bacillus subtilis natto OK2 strain. The OK2 strain would only cross-feed with the Escherichia coli bioB mutant and also grew well in medium containing dethiobiotin. Sequencing analysis revealed two significant genetic alterations in the bioW and bioF genes within the bio operon of the OK2 strain. Complementation analysis with B. subtilis 168 bio mutants demonstrated that only the bioB gene could complement, but other bio operon genes could not. A bio+ transformant, isolated from an OK2 strain, has biotin autotrophy.
The phenylacetic acid (PhAc) degradation pathway becomes an interesting model for the catabolism of aromatic compounds. To determine the molecular basis for this environmentally important process, we did a phylogenic analysis based on the PhAc CoA ligase gene. It suggests that the PhAc CoA ligase genes are distributing widely and subject to frequent lateral gene transfer within and across bacterial phylum.
Insulin microcrystals were encapsulated (microcrystal/PLGA) within poly(lactide-co-glycolide) (PLGA 50:50) by the multiple emulsification solvent evaporation technique and compared with insulin solution microspheres (solution/PLGA) in terms of their morphology, size distribution, drug content, encapsulation efficiency, and stability of insulin during release.
We cloned the gene and corresponding cDNA for an extracellular Rhodotorula-lytic enzyme which has β-1,3-mannase activity, tentatively named MAN5C, from Paecilomyces lilacinus. MAN5C showed a high homology score with the members of glycoside hydrolase family 5 in a domain search with the Pfam database, indicating that MAN5C is a novel and unique member of glycoside hydrolase family 5.
The effect of maternal protein restriction during pregnancy on the offspring’s blood pressure was assessed in stroke-prone spontaneously hypertensive rats (SHRSP) which are genetically predisposed to hypertension and stroke. After the confirmation of pregnancy, the control group was given a 20% casein diet, and the low-protein group was fed a 9% casein diet. After the confirmation of delivery, commercial feed was given to both of the groups. No differences were seen between the control and low-protein offspring in regard to body weight, blood pressure elevation, or life span. One percent saline solution was put in the control and low-protein groups after the age of 11 weeks. Blood pressure increased markedly in the low-protein group, on the blood pressure level in the low-protein group on week 2 after salt loading (242±6 mmHg) was significantly higher than that in the control group (223±9 mmHg; p<0.05). The survival duration was significantly shorter in the low-protein group (113±4 days) than in the control group (135±22 days; p<0.05). These results suggest that maternal protein malnutrition in SHRSP exerted a high salt sensitivity and a malignant influence on stroke incidence on offspring.
Efficiency was examined of microencapsulating L-ascorbic acid by polyglycerol monostearate (PGMS), and changes in the chemical and sensorial aspects of L-ascorbic acid and/or iron-fortified milk during storage were evaluated. The selected core materials were ferric ammonium sulfate and L-ascorbic acid. The highest efficiency (94.2%) of microencapsulation was found with the ratio of 5:1 as the coating to core material. The release of ascorbic acid from the microcapsules increased sharply from 1.6 to 6.7% up to 5 d of storage. The TBA value was the lowest in the milk sample with added encapsulated iron and unencapsulated L-ascorbic acid up to 5 d of storage in comparison with the other treated samples. A sensory analysis showed that most aspects were not significantly different between the control and fortified samples encapsulated with ascorbic acid after 5 d of storage. The results indicate that L-ascorbic acid microencapsulated with PGMS can be applied to fortify milk and acceptable milk products can be prepared with microencapsulated L-ascorbic acid and iron.
Stored cut lettuce gradually turns brown on the cut section after several days of storage, because cutting induces phenylalanine ammonia-lyase (PAL) activity, the biosynthesis of polyphenol is promoted, and the polyphenols are oxidized by polyphenol oxidase. Here, the effect of heat shock treatment at 50°C for 90 s on the quality of cut lettuce during cold storage was examined. The heat shock treatment significantly repressed the induction of PAL activity and phenolics accumulation in cut lettuce during storage, and prevented the browning of cut lettuce. Ascorbic acid content was not affected by the heat shock treatment. The sensory analysis showed that the organoleptic quality of cut lettuce treated by heat shock was significantly better than that of the control cut lettuce. These results show that heat shock treatment is useful for prolonging the shelf life of cut lettuce.
Modulation of the activity and content of cytochrome P-450 (CYP) in hepatic microsomes may be important to human health since these enzymes activate and inactivate a wide range of xenobiotics and food components. Regulation of the inducibility of most CYPs involves transcriptional regulation and post-transcriptional mRNA stabilization. We examined in the present study the effect of dietary soy isoflavone (0–300 mg of isoflavone/kg of diet) on the mRNA abundance of rat hepatic CYP1A1, CYP1A2, CYP2B1/2, CYP2C11, CYP2E1, CYP3A1, CYP3A2 and CYP4A1 by quantitative competitive RT-PCR and real-time monitored RT-PCR. A fermented soy extract containing 155 mg/g of genistein, 127 mg/g of daidzein, and other minor isoflavones was used as the isoflavone source. The dietary soy isoflavone had no affect on the hepatic mRNA abundance of these CYPs. The results by both methods were well matched and indicate that the dietary soy isoflavone did not cause the induction of CYPs by transcriptional step-up regulation or post-transcriptional mRNA stabilization.
A model solution of orange juice containing sugars, ascorbic acid, and citric acid was prepared and its browning during storage was examined. The solution gradually turned brown. Ascorbic acid (AsA) most contributed to the browning. Citric acid and such amino acids as Arg and Pro promoted the browning. DTPA, a strong chelator, inhibited the browning. 3-Hydroxy-2-pyrone (3OH2P), 5-hydroxymethylfurfural (HMF), furfural, 5-hydroxymaltol, and 2-furoic acid were identified as decomposed products in the stored solution. When 3OH2P was stored, the solution turned slightly brown. Furfural solution added with amino acids turned yellow. 3OH2P showed a positive relation with the browning of retail orange juice during storage.
Lactoferrin (LF), a multifunctional milk protein, is known to inhibit in vitro infection by viruses such as herpes simplex virus type 1 (HSV-1). We evaluated the influence of LF feeding on the HSV-1 cutaneous infection of mice. Bovine LF was administered to mice and, after 10 d, the mice were infected with HSV-1. LF administration did not affect the viral clearance in the skin, but inhibited the appearance of skin lesions. LF prevented body weight loss and the decrease of splenocyte number associated with HSV-1 infection. LF increased the serum interleukin (IL)-18 level and splenocyte production of interferon-γ and IL-12. These results suggest that LF feeding was not effective for eradication of the virus, but may contribute to the maintenance of homeostasis and the concomitant increases of cytokine responses during HSV-1 infection.
Vinegar was produced from barley-, sweet potato-, and rice-shochu post-distillation slurry using jar fermentor within 19 hrs. All the vinegars showed radical-scavenging activity, angiotensin I converting enzyme (ACE) inhibition and advanced glycation endproducts (AGE) inhibition in vitro. The radical-scavenging activity of the vinegar produced from sweet potato-shochu post-distillation slurry was higher than that of other two kinds of vinegar on the organic matter basis. The ACE inhibitory activities of all the vinegars were higher than that of each post-distillation slurry. The main components that showed ACE inhibitory activity would be peptides, and their content increased during acetic acid fermentation. Regarding AGE inhibition, only rice-shochu post-distillation slurry did not show such activity, but the other two post-distillation slurries and all the vinegars showed clear inhibitory activity. The activity appeared to depend on the concentration of amino groups except for sweet potato-shochu post-distillation slurry and the vinegar produced from it.
Transferrin was isolated and purified from bovine plasma. An intestinal segment in situ experiment showed that 19.2% of injected iron was absorbed when FeCl3 (80 μg Fe/ml) was injected into a duodenum segment of iron-deficient rats. With addition of 10 and 20 mg of purified transferrin/ml, however, ratios of absorbed iron through duodenum segments were significantly increased to 52.7 and 57.9%, respectively. After transferrin-rich extract was isolated by batch type ion exchange chromatography, a soluble ferric complex of the transferrin extract was prepared by adding ferric salts to transferrin extract followed by dialysis, sterilization, and freeze drying. Results of the animal experiment for comparing bioavailabilities of different irons showed that irons in Fe-transferrin extract was most efficiently absorbed and incorporated into hemoglobin generation in anemic rats.
Interest in the beneficial effects of polyphenols, including tannic acid (TA), is increasing, although, these compounds also have adverse effects; for example, on the absorption of iron (Fe), and possibly other trace minerals. We examined the effect of a graded dose of TA on the absorption of Fe and compared with that of zinc (Zn), copper (Cu) and manganese (Mn) in rats. We also investigated the effect of TA on cecal fermentation which plays a role in absorption. In Experiment 1, to set the optimum dose of Fe, male Sprague-Dawley rats (weighing 70–90 g) after acclimatization were fed with different levels of dietary Fe (5, 10, 20, 30 and 35 mg/kg). We observed that the hematocrit (Ht), serum Fe concentration and transferrin saturation (%) were each reduced in those rats fed less than 20 mg/kg Fe in a dose-dependant manner. In Experiment 2, the rats were fed with test diets containing the minimum required level of Fe, 30 mg/kg diet, with (5, 10, 15 and 20 g/kg diet) or without TA for a period of three weeks. Feeding a diet containing more than 10 g TA/kg diet, but not 5 g TA/kg diet, reduced the hemoglobin concentration (Hb), Ht and serum Fe concentration due to decreased Fe absorption. In contrast, the Zn, Cu and Mn absorption was not affected by TA feeding. It is also demonstrated that liver Fe, but not the Zn, Cu and Mn contents, were lower in the TA groups than in the TA-free control group. Feeding TA slightly decreased the pH value of the cecal contents with an increase in the major short-chain fatty acid pool. About 15% of the ingested TA were recovered in the feces of each TA-fed group. Our results demonstrate that more than 10 g TA/kg diet induced anemia by reducing the Fe absorption, although there was no effect on the absorption of other important trace minerals. Our findings suggest that the usual intake of polyphenols is relatively safe, but that a high intake by supplementation or by dietary habit of tannin affects only the Fe level.
One- and two-dimensional 1H nuclear magnetic resonance (1H NMR) spectra were measured in order to estimate the dissociation constants (Kdiss) and molecular geometries of cyclodextrin (CD) with three organic isothiocyanates (ITC), allyl-ITC, 3-butenyl-ITC, and 4-pentenyl-ITC, in an aqueous solution (pH 5.0, Ic=0.75 M). In every ITC, the Kdiss values decreased in the order of α-CD>β-CD>γ-CD, and the three rod-like linear molecules of ITCs were accommodated with the best fit into the smallest cavity of α-CD. By rotating-frame nuclear Overhauser enhancement spectroscopy, cross peaks were found between the H-3 of α-CD and the H-1 of AITC. From the result, the molecular geometry of α-CD–AITC complex presume that the isothiocyanate group of AITC is located some where around the wideing rim and a hydrophobic allyl group is included within the hydrophobic cavity of α-CD.
Ethyl 4,7,10,13,16,19-docosahexaenoate (DHA-Et) dissolved in an ethanol solution or embedded in liposomes was degraded by irradiating with γ rays in a dose-dependent manner. The degradation rate of DHA-Et embedded in liposomes was higher than that of DHA-Et dissolved in ethanol. Antioxidants suppressed the degradation of DHA-Et embedded in liposomes, the order of activity of the antioxidants being luteolin>fisetin>kaempferol>quercetin>rutin. These results suggest that the hydrophobicity (logP) of an antioxidant is one of determinants for antioxidative activity, but that a vicinal diol structure in the B ring is not favorable for the antioxidative activity.
The activities of NAD-independent D- and L-lactate dehydrogenases (D-LDH, L-LDH) were detected in Rhodopseudomonas palustris No. 7 grown photoanaerobically on lactate. One of these enzymes, D-LDH, was purified as an electrophoretically homogeneous protein (Mr, about 235,000; subunit Mr about 57,000). The pI was 5.0. The optimum pH and temperature of the enzyme were pH 8.5 and 50°C, respectively. The Km of the enzyme for D-lactate was 0.8 mM. The enzyme had narrow substrate specificity (D-lactate and DL-2-hydroxybutyrate). The enzymatic activity was competitively inhibited by oxalate (Ki, 0.12 mM). The enzyme contained a FAD cofactor. Cytochrome c2 was purified from strain No. 7 as an electrophoretically homogeneous protein. Its pI was 9.4. Cytochrome c2 was reduced by incubating with D-LDH and D-lactate.
For functional analysis, the presence of gene families and isoenzymes often makes it necessary to delete more than one gene, while the number of marker genes is limited in Schizosaccharomyces pombe. Here we describe a loxP-flanked ura4+ cassette and Cre recombinase vector for a Cre-loxP-mediated marker removal procedure in S. pombe. This loxP-ura4-loxP cassette can be used for disruption of hmt1+ as a model target gene. We have constructed two vectors which express Cre recombinase under the control of the nmt1 or nmt41 promoter. Excisive recombination at loxP sites in the chromosome was promoted efficiently and accurately when the Cre recombinase was expressed under the control of the nmt41 promoter. In addition, ura4+ could be excised from the genome by Cre recombinase, when a single loxP site was adjacent to ura4. The use of the Cre-loxP system proved to be a practical strategy to excise a marker gene for repeated use in S. pombe.
Two bacterial strains, 127W and T102, were isolated from anoxic crude oil tank sludge as effective degraders of dibenzothiophene (DBT), a model sulfur containing heterocyclic aromatic compound in crude oil. Strain 127W was more tolerant to oxygen limitation than T102 and was capable of degrading two- and three-ring polycyclic and heterocyclic aromatic compounds under both aerobic and low oxygen conditions. Strain 127W degraded 0.082, 0.055, and 0.064 mM of DBT, naphthalene, and anthracene, respectively, in one week with dissolved oxygen <⁄=0.2ppm (0.006 mM). Degradation by 127W cell-free extracts for DBT was increased by addition of sodium hydrogencarbonate under this oxygen concentration. Phylogenetic analysis of the 16S rRNA gene sequence and physiological characteristics indicate that the strains 127W and T102 belong to new species of the genus Xanthobacter and Pseudomonas stutzeri, respectively. We propose X. polyaromaticivorans sp. nov. 127W.
The Clostridium stercorarium F-9 xyl43B gene encoding the β-xylosidase Xyl43B consists of an open reading frame of 1,491 nucleotides that encodes a putative protein, classified in family 43, of 497 amino acids with a predicted molecular weight of 56,355. The deduced amino acid sequence of Xyl43B has sequence similarity with β-xylosidases from Bacteriodes thetaiotaomicron (57% sequence identity), Prevotella ruminicola (45%), Streptomyces coelicolor (40%), and Clostridium acetobutylicum (36%), all of which have been classified in family 43 of the glycoside hydrolases. Xyl43B was purified from a recombinant Escherichia coli and characterized. The optimum pH of the purified enzyme was 3.5 and it was stable over pH from 3.0 to 8.0. Its optimum temperature was 80°C and it showed thermostability in the temperature range from 50 to 70°C. Xyl43B had a Km of 6.2 mM and a Vmax of 15 μmol min−1 mg−1 for p-nitrophenyl-β-D-xylopyranoside.
Delftia acidovorans MC1 was continuously cultivated under nutristat conditions with elevated concentrations of the herbicides (RS)-2-(2,4-dichlorophenoxy)propionate [(RS)-2,4-DP] and 2,4-dichlorophenoxyacetate (2,4-D). The presence of 1–5 mM of either of these compounds did not essentially inhibit growth. Moreover, substrate consumption was not essentially affected at pH values of 7.0–9.0 selected by reason of alkaline in situ conditions found e.g. on contaminated building rubble but was decreased at pH 9.3. The adenylate energy charge declined to some degree as the herbicide concentration rose, the extent of this increasing as the pH rose. This was caused by an increase in the concentration of ADP and in particular AMP, in contrast to the fairly constant ATP level of around 4 nmol/mg dry mass with (RS)-2,4-DP and 2 nmol/mg with 2,4-D. Comparison of the individual growth parameters with theoretical data taking into account maintenance coefficients of 0.48 mmol (RS)-2,4-DP/g*h and 0.6 mmol 2,4-D/g*h revealed that the culture followed purely kinetic rules. This excludes the necessity of using substrate to a significant extent to satisfy extra efforts in energy for homeostasic work under these accentuated conditions.
To compare NADH-regeneration systems for the synthesis of (S)-4-chloro-3-hydroxybutanoate (ECHB), a novel NADH-dependent carbonyl reductase (KaCR1), which reduced ethyl 4-chloroacetoacetate (ECAA) to form (S)-ECHB, was screened and purified from Kluyveromyces aestuarii and a gene encoding KaCR1 was cloned. Glucose dehydrogenase (GDH) and formate dehydrogenase (FDH) were compared as enzymes for NADH regeneration using Escherichia coli cells coexpressing each enzyme with KaCR1. E. coli cells coexpressing GDH produced 45.6 g/l of (S)-ECHB from 50 g/l of ECAA and E. coli cells coexpressing FDH, alternatively, produced only 19.0 g/l. The low productivity in the case of FDH was suggested to result from the low activity and instability of FDH.
adeA and adeB genes homologous to Saccharomyces cerevisiaeADE1 and ADE2, respectively, were cloned from Aspergillus oryzae. AdeA and AdeB share 62.8% and 52.5% identities with S. cerevisiae Ade1 and Ade2, respectively. In order to obtain triple auxotrophic mutants from A. oryzae, 12 red-colored mutant colonies were isolated by UV mutagenesis of a double auxotrophic host, NS4 (niaD−, sC−), as a parent strain. All the mutants exhibited adenine auxotrophy and showed fluorescence in the vacuoles due to accumulation of a purine biosynthetic pathway precursor. Adenine auxotrophy of all the mutants was restored by introduction of either A. oryzaeadeA or adeB genes. Sequence analysis demonstrated that substitutions or deletions of a single base pair occurred, inducing substitutions or frame shifts of amino acid sequences in both ade genes complementing the mutants. This study provides a novel host-vector system with triple auxotrophy in A. oryzae.