1,1-Dipehnyl-2-picrylhydrazyl (DPPH) radical scavenging activities were found in the extract of dried leaves of oregano (Origanum vulgare). The water-soluble active ingredients were isolated, and their structures were determined to be 4′-O-β-D-glucopyranosyl-3′,4′-dihydroxybenzyl protocatechuate and 4′-O-β-D-glucopyranosyl-3′,4′-dihydroxybenzyl 4-O-methylprotocatechuate by 1H-, 13C-NMR, DEPT, HMQC, and HMBC spectral analyses, and by NOE experiments. The DPPH radical scavenging activities of these compounds were compared with those of rutin, quercetin and rosmarinic acid at a concentration of 2×10-5 M. The scavenging activity of 4′-O-β-D-glucopyranosyl-3′,4′-dihydroxybenzyl protocatechuate was almost the same as that of quercetin and rosmarinic acid, but that of 4′-O-β-D-glucopyranosyl-3′,4′-dihydroxybennzyl 4-O-methylprotocatechuate was less than that of quercetin, rosmarinic acid and 4′-O-β-D-glucopyranosyl-3′,4′-dihydroxybenzyl protocatechuate. The amount of 4′-O-β-D-glucopyranosyl-3′,4′-dihydroxybenzyl protocatechuate was estimated to be 3.8 mg/1 g of dried leaves by an HPLC analysis.
The concentration of abscisic acid in plants is regulated not only by biosynthesis, but also by metabolism. Abscisic acid is metabolized to phaseic acid via 8′-hydroxyabscisic acid, and phaseic acid is then converted to dihydrophaseic acid and its epimer. A quantitative analysis of these metabolites is important as well as that of abscisic acid to understand changes in the concentration of abscisic acid in plants. However, no internal standards of the metabolites suitable for quantitative analysis have been reported. We prepared 7′-deuterium-labeled phaseic acid with a deuterium content of 86%, using the equilibrium reaction between phaseic acid and 8′-hydroxyabscisic acid. 7′-Deuterium-labeled dihydrophaseic acids were obtained by reducing 7′-deuterium-labeled phaseic acid. The levels of the metabolites in plant organs were determined by using the deuterated metabolites as internal standards.
4-[2-(3,5-Dimethoxyphenyl)ethenyl]-1,2-benzenediol (7), a stilbene isolated from Sphaerophysa salsula, was synthesized from 3,4-dihydroxybenzaldehyde (1) in five steps in an overall yield of 33%. The spectral data for synthetic 7 are in good agreement with those of the natural product. Hydroxystilbene 7 showed potent antioxidative activity.
Telomerase is a ribonucleoprotein complex the function of which is to add telomeric repeats (TTAGGG)n to chromosomal ends, and it is known to play an important role in cellular immortalization. Telomerase is highly active in most tumor cells, yet not in normal cells. As such, it may have possible applications in cancer gene therapy. Telomerase consists of two essential components, telomerase RNA template (hTR) and catalytic subunit (hTERT). hTERT is expressed only in cells and tissues positive for telomerase activity, i.e., tumor and fetal cells. We here tested the possibility of the utilization of the hTERT promoter in targeted cancer gene therapy. We cloned the hTERT promoter in the replace of the CMV promoter and sub-cloned HSV-TK gene to be controlled by hTERT gene promoter in adenovirus shuttle plasmid. Then we constructed recombinant adenovirus Ad-hT-TK, and infected them into normal and human gynecological cancer cell lines. Through these experiments, we identified the selective tumor specific cell death by Ad-hT-TK. Furthermore, FACS analysis and TUNEL assay suggests that the reduced viability is mediated through the induction of apoptosis, indicating that this approach may be a useful method for suppressing cancer growth in targeted cancer gene therapy. These results show that Ad-hT-TK could be used for gynecological cancer gene therapy.
A putative α-glucosidase belonging to glycosyl hydrolase family 4 of Thermotoga maritima (TM0752) was expressed in Escherichia coli and it was found that the recombinant protein (Agu4B) was a p-nitrophenyl α-D-glucuronopyranoside hydrolyzing α-glucuronidase, not α-glucosidase. It did not hydrolyze 4-O-methyl-D-glucuronoxylan or its fragment oligosaccharides. Agu4B was thermostable with an optimum temperature of 80°C. It strictly required Mn2+ and thiol compounds for its activity. The presence of NAD+ slightly activated the enzyme. The amino acid sequence of Agu4B showed higher identity with Agu4A (another α-glucuronidase of T. maritima, 61%) than with AglA (α-glucosidase of T. maritima, 48%).
Kalihinol F, a naturally occurring diterpene from a marine sponge, Acanthella sp., inhibited chromosome separation in fertilized starfish (Asterina pectinifera) eggs but allows the first cleavage to occur, thereby forming unseparated metaphase chromosomes which were elongated between the two daughter cells. The chromosomes were eventually torn off in the embryonic cells. Most of the cells gradually lost the chromosomes during the cell cycle progression. The embryonic development halted at the morula stage just before the onset of blastulation. The mitotic failure occurred when kalihinol F was applied to a fertilized egg during the second meiotic process, but not after the completion of the second meiotic division. Kalihinol F inhibited topoisomerase I activity in vitro, but had no effects on activities of DNA polymerases α, β, and γ, and of topoisomerase II. These results suggest that the topoisomerase I plays an essential role in meiosis II in this species.
Replication factor C (RFC) catalyzes the assembly of circular proliferating cell nuclear antigen (PCNA) clamps around primed DNA, enabling processive synthesis by DNA polymerase. The RFC-like genes, arranged in tandem in the Thermococcus kodakaraensis KOD1 genome, were cloned individually and co-expressed in Escherichia coli cells. T. kodakaraensis KOD1 RFC homologue (Tk-RFC) consists of the small subunit (Tk-RFCS: MW=37.2 kDa) and the large subunit (Tk-RFCL: MW=57.2 kDa). The DNA elongation rate of the family B DNA polymerase from T. kodakaraensis KOD1 (KOD DNA polymerase), which has the highest elongation rate in all thermostable DNA polymerases, was increased about 1.7 times, when T. kodakaraensis KOD1 PCNA (Tk-PCNA) and the Tk-RFC at the equal molar ratio of KOD DNA polymerase were reacted with primed DNA.
We confirmed that cholesterol esterase accelerated the incorporation of unesterified cholesterol solubilized in bile salt micelles into differentiated Caco-2 cells under various experimental conditions. Rat pancreatic juice and bovine cholesterol esterase increased the incorporation of micellar cholesterol into rat intestinal brush border membranes. The incorporation of micellar cholesterol was not changed in the brush border membranes enriched in and depleted of cholesterol esterase. The results suggest that the accelerated incorporation of micellar cholesterol by cholesterol esterase into absorptive cells is not mediated by the enzyme bound to the brush border membranes.
The effects of protease inhibitors on the production of recombinant protein were investigated using a recombinant baculovirus containing GFPuv-human β1,3-N-acetylglucosaminyltransferase 2 (β3GnT2) connected to the prepromelittin signal sequence. The addition of leupeptin as a cysteine protease inhibitor at 2.5 μg/ml improved intra- and extracellular β3GnT activities 5- and 3-fold, respectively, compared to those without addition, which was due to a suppression of protease activity. With the leupeptin addition only four degraded molecular bands lower than 32 kDa appeared, but 9 degraded molecular bands between 29 kDa and 41 kDa existed without addition. In contrast, pepstatin A as a carboxyl protease inhibitor had no influence on the improvement of β3GnT production, judging from SDS-PAGE. Moreover, when 50 μM carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG-132), known as a proteasome inhibitor, was used in combination with the leupeptin, a ladder of low molecular mass bands of fusion protein was diminished. The intracellular β3GnT activity increased 9-fold, to as high as that without addition of two kinds of protease, but the extracellular activity was not different from that with the addition of only leupeptin. These findings indicate that the decrease in cell viability causes the decrease in the secretion rate of intracellular fusion protein, resulting the accumulation of the full-length of fusion protein.
Morphological and functional changes of rat pancreatic islets caused by administration of streptozotocin (STZ) and the bioavailability of insulin formulations administered to STZ-induced diabetic rats with fasting (12 h) or non-fasting were investigated. Islets isolated from normal rats maintained a good three-dimensional structure and the islet yield was 962.5±86.5 islet equivalent number (IEQ, islets converted to an average diameter of 150 μm). In the diabetic group (>500 mg/ml blood glucose), the islet yield was only 44.4±8.3 IEQ and the islet was severely damaged. The minimum reduction of blood glucose of each formulation, such as insulin solution, microcrystal, and insulin microcrystal capsule, was shown to be 11.3, 11.0, and 16.3 mg/dl, respectively, at 6 h in fasting with diabetic rats. These results indicated that the administration of insulin formulations to the fasting groups increased the severe hypoglycemic effect of insulin action more than in non-fasting diabetic rats. The diabetic rat with fasting has a regulatory disorder in maintaining the blood glucose level. Accordingly, the validity of pharmacological availability as an optimal modeling of insulin formulations is best in non-fasting STZ-induced diabetic rats.
The chitin-binding domain of human macrophage chitinase was expressed as a fusion protein with glutathione S-transferase in Escherichia coli and assayed for its binding activity. The purified recombinant chitin-binding domain bound to chitin, but not to glucan, xylan, or mannan. The binding of the recombinant chitin-binding domain to chitin was inhibited by N-acetylglucosamine, di-N-acetylchitobiose, and hyaluronan, but not by N-acetylgalactosamine or chondroitin. Furthermore, a solid-phase binding assay showed that the recombinant domain interacts specifically with hyaluronan and hybrid-type N-linked oligosaccharide chains on glycoproteins, and that the oligosaccharide-binding characteristics are similar to those of wheat germ agglutinin, a lectin that binds to chitin. The results suggest that human chitinase chitin-binding domain may be involved in tissue remodeling through binding to polysaccharides or extracellular matrix glycoproteins, and this recombinant protein can be used to elucidate biological functions of the enzyme.
A gene encoding a Δ9 fatty acid desaturase (Δ9-desaturase) was isolated from L. edodes. The open reading frame of this gene (named Le-FAD1) consisted of 1416 bp. The predicted protein of 471 amino acids shares 47-57% sequence identity with Δ9-desaturases from other fungi. Expression of the Le-FAD1 ORF complemented genetic disruption of the Saccharomyces cerevisiaeOLE1 gene (Sc-OLE1). Fatty acid analysis of the Le-FAD1-expressing S. cerevisiaeole1-disrupted strain showed that Le-FAD1 protein had slight activity with palmitic acid (16:0) as the substrate and high activity with stearic acid (18:0). Analysis of transcriptional expression showed that the levels of Le-FAD1 mRNA in the primordium and fruiting body of L. edodes were higher than in mycelia cultivated at 18°C as well as 25°C. These results correlated with the increase of unsaturated fatty acids (UFAs) in total lipids during fruiting-body formation.
A GA biosynthesis inhibitor, uniconazole, caused many shrunken embryos when it was supplied to cultured carrot (Daucus carota L.) cells at the induction of somatic embryos. The abnormality was prevented by exogenous GA1 or GA4. To analyze the status of GA biosynthesis during somatic embryogenesis, expression patterns of newly isolated genes encoding GA biosynthetic enzymes, two GA 20-oxidases, three GA 3-oxidases, and two GA 2-oxidases were observed by using a semi-quantitative reverse-transcription-polymerase chain reaction with gene-specific primers. Transcript levels of GA 20-oxidases and GA 2-oxidases did not change greatly during development of the somatic embryo. On the other hand, drastic changes were found in three GA 3-oxidase genes. Strikingly, expression of a GA 3-oxidase gene, DcGA3ox2, was elevated once in somatic embryogenesis, but not in the non-induced suspension cells. The enzymatic functions of these gene products were also confirmed using recombinant proteins expressed in Escherichia coli. Our results indicate that GA biosynthesis is required for carrot somatic embryogenesis.
We examined the effects of serum deprivation on myofibrillar proteolysis in chick myotubes. Myotubes were incubated with serum-free medium for 24 hours. Nτ-methylhistidine release, as an index of myofibrillar proteolysis, as well as protease activities such as calpain, proteasome, and cathepsins (B+L and D) activities were increased by serum deprivation. These results indicate that serum deprivation induces calpain, proteaosme, and cathepsins activities, resulting in an increase in myofibrillar proteolysis in chick myotubes.
Increased blood-DNA breakage was observed in diseased pearl oysters. They showed significant formation of 8-hydroxydeoxyguanosine (8-OHdG) and malondialdehyde (MDA), whereas the oysters that had a low mortality rate from the disease had high activity of superoxide dismutase (SOD) and low amounts of 8-OHdG and MDA. These results suggest that radical damage had occurred only in the diseased pearl oysters with the cytolysis of their haemocytes, which was related to the mass mortality of the Japanese pearl oysters.
An improvement in the method of the Reverse Transcription PCR (RT-PCR) using RNase H is proposed here. We succeeded in RT-PCR amplification against the full sequence of the coding region (8.9 kb) of the Insulin-like growth factor II receptor gene which has the area called the GC-block of about 90% GC contents at the 5′ terminal. Furthermore, the RNase H treatment improved the sensitivity of RT-PCR amplification against a general target.
We report here for the first time on the construction of proteomes from wheat lemma at the anthesis stage. After transfer of lemma proteins to polyvinylidene difluoride membranes, seventy larger spots were subjected to peptide sequence analysis; the amino acid sequences could be described for forty-eight of these proteins. The result suggested that wheat proteins were less N-terminally blocked compared to rice proteins, which are known to have a much higher ratio of N-terminal blocks. We further analyzed the internal sequences of eight blocked proteins by the Cleveland peptide mapping method. Out of these total 56 amino acid sequences, forty-one could be assigned to the corresponding expressed sequence tags (ESTs). The expression profile of lemma proteins was generally similar to that of leaf, and the majority of identified proteins were related to cellular metabolisms. We analyzed the internal sequences of one protein spot present in lemma, which was not present in leaf.
Transglutaminase 3 (TGase 3), involved in the cross-linking of structural proteins in the epidermis, is activated by limited proteolysis of zymogen into two fragments during keratinocyte differentiation. Using recombinant TGase 3, the N-terminus sequence of the proteolyzed fragment was analyzed. Antibody against the synthetic peptide corresponding to the cleavage site specifically detected the fragment in the mouse forestomach extract.
Arabidopsis ZIM is a putative transcription factor containing an atypical GATA-type zinc-finger motif. Transcriptional activation by ZIM was tested using a transient GAL4 fusion assay and measuring the expression of a luciferase reporter in tobacco BY-2 cells. ZIM functioned as a transcriptional activator, and the transactivation domain was found to occur in its N-terminal acidic region.
The transepithelial transport of such common dietary phenolic acids as p-coumaric acid (CA) and gallic acid (GA) across Caco-2 cell monolayers was examined. CA transport was dependent on pH, and in a vectorial manner in the apical-basolateral direction. The permeation was concentration-dependent and saturable, the Michaelis constant and maximum velocity being 17.5 mM and 82.7 nmol min-1 (mg of protein)-1, respectively. Benzoic acid and acetic acid inhibited the permeation of CA. These results indicate that the transepithelial transport of CA was via the monocarboxylic acid transporter (MCT). On the other hand, the permeation of GA was not in a polarized manner, was independent of pH and linearly increased with increasing concentration of GA. The transport rate of GA was about 100 times lower than that of CA, suggesting the transepithelial transport of GA to be via the paracellular pathway. Dietary phenolic acids thus showed diversified characteristics in their intestinal absorption.
We assessed the effects of intake of thiamin, arginine, caffeine, and citric acid (TACC) on lipid metabolism in healthy subjects. Thirty-one subjects with high percent body fat (≥25.0%) were randomly assigned to a 12-wk intervention with daily intake of TACC-supplemented tea (1.1, 1240, 52, and 540 mg, respectively; n=16) or control tea (n=15). The percent body fat decreased significantly during the intervention in both groups, especially in the TACC group. A percentage decrease in triceps skinfold was significantly greater in the TACC group than in the control group. The decrease in abdominal visceral fat in obese subjects was significantly greater in the TACC group than in the control group. Serum triglyceride was significantly lower during intervention than that during the non-intervention period in the TACC group. These results suggest that TACC may be effective in reducing body fat in obese subjects.
Various mutant lysozymes having the N-glycosylation signal sequence, R21T (Asn19-Tyr20-Thr21), G49N (Asn49- Ser50-Thr51), R21T/G49N (Asn19-Tyr20-Thr21/Asn49-Ser50-Thr51), were secreted in the Pichia pastoris expression system. The secreted amounts of these mutant glycosylated lysozymes were almost the same as those of wild-type lysozyme (about 30 mg/liter). Glycosylation of the mutant lysozymes was confirmed by SDS-PAGE patterns, Endo-H treatment, TOF-MS analysis and chemical analysis. The composition of the carbohydrate chain attached to the single glycosylated lysozymes, R21T and G49N, was GlcNAc2Man9-11, while that of the double glycosylated lysozyme, R21T/G49N, was GlcNAc4Man27-32. The results of a CD analysis and lytic activity suggested that the conformation of the single glycosylated lysozymes had been conserved, while that of the double glycosylated lysozyme was less stable. The emulsifying properties of the lysozyme when glycosylated were greatly improved, being especially noteworthy in the double glycosylated lysozyme.
An attempt was made to mask the allergenic structure of a major allergen protein, Cry j I (CJI), in Japanese cedar pollen using the Maillard-type polysaccharide conjugation. The SDS-PAGE pattern of the CJI-galactomannan conjugate prepared by the Maillard reaction showed broad bands widely distributed from 50 kDa to more than 100 kDa, suggesting the attachment of galactomannan. The competitive enzyme-linked immunosorbent assay showed that the IgE antibody in the sera of ceder pollen-sensitive patients reacted strongly with CJI, while it did not react with the CJI-galactomannan conjugate. This result suggests that the antigenicity of CJI is greatly reduced by the conjugation with galactomannan.
We studied lipid metabolism to evaluate the effects of γ-terpinene on suppression of increases in serum lipid concentrations using Triton WR1339-treated rats. At 6 hr after Triton WR1339 injection, the total cholesterol and triglyceride concentrations in the γ-terpinene group underwent statistically significant decreases (18.3 and 30.3%, respectively) compared with those of the Triton-treated group.
These experiments were done to clarify that the differential effects of thyroxine (T4) and triiodothyronine (T3) on skeletal muscle protein turnover are caused by their roles on ATP production. Primary cultured chick muscle cells were treated with a physiological level of T4 (60 ng/ml), T3 (12 ng/ml), or ATP (0.5 mM) for 6 days and the protein content, ATP production, proteasome activity, and myofibrillar protein breakdown were measured. The protein content measured as an index of cell growth was not affected by T4, T3, or ATP. The cellular ATP level was increased by T3 and ATP, but not by T4. Proteasome activity and Nτ-methylhistidine (MeHis) release measured as an index of myofiblillar protein breakdown was also increased by T3 and ATP, but not by T4. These results indicate that T3 but not T4 increases ATP production followed by an increase in proteasome activity, and thus stimulates myofibrillar proteolysis.
The combined use of a fluorescent glucose (2NBDG) and a fluorescent glycine (NBD-Gly) was tried for the detection of viable cells of significant foodborne pathogenic strains in addition to several Escherichia coli strains and coliforms. Thirty-five out of 41 strains showed marked uptake of 2NBDG but 6 strains were not able to take in 2NBDG. Five out of these 6 strains showed NBD-Gly uptake.
Beta-Cryptoxanthin (βCx) was investigated for cell functions in neuroblastoma Neuro2a cells. The following results were obtained. 1. Beta-Cx induced neurite outgrowth. 2. Beta-Cx inhibited the etoposide-induced activation of caspase-3 activity in a dose-dependent manner. These data suggest a bioregulatry function of βCx in the control of differentiation and apoptosis in Neuro2a cells.
Porphyra yezoensis (Susabinori, an edible purple laver), which was cultured aseptically for 12 weeks and then lyophilized, contained 50±2 μg/g of vitamin B12 per 100 g dry weight. Coenzyme forms of vitamin B12 (about 60% of the total vitamin B12) were found in the cultured purple laver aseptically, which may have the ability to biosynthesize the coenzymes.
Increase in plasma immunoglobulin G specific to orally administered ovalbumin in Brown Norway rats was retarded by feeding enzyme-treated wheat flour when compared with untreated flour. Because plasma ovalbumin concentrations after feeding ovalbumin tended to be lower in mice fed enzyme-treated flour than in those fed untreated flour, suppression of ovalbumin absorption may be relevant to retarded sensitization observed in rats.
Trp, Phe, and Tyr ethyl esters and their dipeptides with Gly at the C-terminals inhibited ovalbumin (OVA) permeation through Caco-2 monolayers. The inhibitory activity of Trp ethyl ester was the highest at near the concentration of 10-6 M. It was suggested that Trp ethyl ester inhibited transcellular permeation of OVA.
The effects of intracerebroventricular (i.c.v.) injection of bovine β-casomorphin-5 (β-CM-5: Tyr-Pro-Phe-Pro-Gly), a μ-opioid agonist derived from milk β-casein, on step-down type passive avoidance tasks were investigated in mice. Intracerebroventricular administration of a high dose (10 μg) of β-CM-5 produced a significant decrease in step-down latency. β-Funaltrexamine (5 μg, i.c.v.) almost completely reversed the β-CM-5-induced shortening of step-down latency, although neither naltrindole (5 ng, i.c.v.) nor nor-binaltorphimine (5 μg, i.c.v.) had any significant influence on the effect of β-CM-5. Meanwhile, a low dose (0.5 μg, i.c.v.) of β-CM-5 inhibited scopolamine (1 mg/kg)-induced impairment of passive avoidance response. These results indicated that a high dose of β-CM-5 induces amnesia, whereas a low dose ameliorates scopolamine-induced amnesia.
Genes encoding an aniline dioxygenase of Frateuria sp. ANA-18, which metabolizes aniline via the ortho-cleavage pathway of catechol, were cloned and named tdn genes. The tdn genes were located on the chromosomal DNA of this bacterium and weren't clustered with catechol-degrading gene clusters. These results show that the ANA-18 aniline-degrading gene cluster is constructionally different from Pseudomonas tdn and Acinetobacter atd gene clusters, which degrade aniline via the meta-cleavage pathway of catechol and organize catechol-metabolic genes in the gene clusters. When cloned tdnQTA1A2B genes were expressed in Eschherichia coli, aniline dioxygenase activity was observed. Southern blot analysis revealed that homologues of the tdnA1A2B genes didn't exist in strain ANA-18. Disruption of the tdnA1A2 genes gave the parent strain ANA-18 a defect in aniline metabolism. On the basis of these results, we concluded that only the cloned tdn genes function as genes encoding aniline dioxygenase in strain ANA-18 although this bacterium had two catechol-degrading gene clusters.
Corynebacterium glutamicum mutant KY9707 was originally isolated for lysozyme-sensitivity, and showed temperature-sensitive growth. Two DNA fragments from a wild-type C. glutamicum chromosomal library suppressed the temperature-sensitivity of KY9707. These clones also rescued the lysozyme-sensitivity of KY9707, although partially. One of them encodes a protein of 382 amino acid residues, the N-terminal domain of which was homologous to RNase HI. This gene suppressed the temperature-sensitive growth of an Escherichia coli rnhA rnhB double mutant. We concluded that this gene encodes a functional RNase HI of C. glutamicum and designated it as rnhA. The other gene encodes a protein of 707 amino acid residues highly homologous to RecG protein. The C. glutamicum recG gene complemented the UV-sensitivity of E. coli recG258::kan mutant. KY9707 showed increased UV-sensitivity, which was partially rescued by either the recG or rnhA gene of C. gluamicum. Point mutations were found in both recG and rnhA genes in KY9707. These suggest that temperature-sensitive growth, UV-sensitivity, and probably lysozyme-sensitivity also, of KY9707 were caused by mutations in the genes encoding RNase HI and RecG.
A glucosylsterol, β-sitosterol-3-O-glucopyranoside, has been isolated as an active principle with sortase inhibitory effect from the bulbs of Fritillaria verticillata by bioassay-guided chromatographic fractionation. The isolate was a potent inhibitor of sortase, with an IC50 value of 18.3 μg/ml and had antibacterial activity against Bacillus subtilis, Staphylococcus aureus, and Micrococcus leuteus with MIC values of 50, 200, and 400 μg/ml, respectively, indicating that this compound is a possible candidate for the development of a bacterial sortase inhibitor. In addition, sitosterol was found to be inactive upon sortase and bacterial cell growth. These results suggest that the inhibitory potency of β-sitosterol-3-O-glucopyranoside is sensitively dependent upon the glucopyranoside side chain moiety.