The four stereoisomers of 7-acetoxy-15-methylnonacosane (1), a component of the female sex pheromone of the New World screwworm fly (Cochliomyia hominivorax) were synthesized. The stereogenic center at C-15 of 1 originated from that of the enantiomers of citronellal, and that at C-7 was generated by lipase-catalyzed asymmetric acetylation of (3RS,11R)- and (3RS,11S)-17-methyl-1-trimethylsilylpentacos-1-yn-3-ol (13). Three of the stereoisomers of 1 showed equivalent good pheromone activity, while the activity of (7R,15R)-1 was weak.
In our search for new cyathane metabolites related to the biosynthesis of erinacine Q in Hericium erinaceum, we isolated a novel cyatha-3,12-dien-14β-ol named erinacol together with known 11-O-acetylcyathatriol (the erinacine Q aglycon) and new metabolite 11-O-acetylcyathin A3 from the mycelial extract. The structure of each compound was determined by spectral methods. Possible biosynthetic relationships of these metabolites are discussed from their structural features.
Telomerase is a ribonucleoprotein complex of which the function is to add telomeric repeats to chromosomal ends. Telomerase consists of two essential components, the telomerase RNA template (hTR) and the catalytic subunit (hTERT). hTERT is expressed only in cells and tissues positive for telomerase activity, i.e., tumor and fetal cells. The aim of this study is to test the increased telomerase promoter activity for cancer gene therapy in adenovirus vector. We cloned the hTERT promoter in place of the SV40 promoter in the pGL3-contol vector to be increased by the SV40 enhancer sequences, resulting in strong expression of luc+ only in telomerase positive cancer cells. Then we transfected the constructed plasmid into a normal human cell line and several cancer cell lines. Through these experiments, we identified the selective and increased expression of the luciferase gene controlled by the hTERT promoter and the SV40 enhancer in the telomerase positive cancer cell lines. To investigate the possibility of utilizing the hTERT promoter and the SV40 enhancer in targeted cancer gene therapy, we constructed an adenovirus vector expressing HSV-TK controlled by the hTERT promoter and the SV40 enhancer for the induction of specific telomerase positive cancer cell death. NSCLC cells infected by Ad-hT-TK-enh were more significantly suppressed and induced apoptosis than those infected by Ad-hT-TK. Telomerase is activated in 80∼90% of cancers, so adenovirus with increasing telomerase promoter activity might be used for targeted cancer gene therapy using suicide genes. These results show that the hTERT promoter and the SV40 enhancer might be used for targeted cancer gene therapy.
Gene targeting to knock out the activity of specific genes has become important due to recent progress in genomics research. But this technique is still unavailable for many organisms, including economically important microorganisms, due to the high background of ectopic integration during genetic transformation. Strategies to improve targeting efficiency have included manipulating the expression of genes that are involved in homologous recombination. In this study, transcription of Aspergillus nidulansuvsC was elevated using the promoter sequences of the glyceraldehyde-3-phosphate dehydrogenase and Taka-amylase A genes from A. nidulans and A. oryzea respectively. Although a several-fold increase in the efficiency of targeting was observed at 3 loci, mycelial growth was suppressed in strains that had higher levels of uvsC transcription. These results suggest that uvsC is a rate-limiting factor in gene targeting, and that the increased efficiency of this targeting is hindered by a negative effect of increased transcription on cell proliferation.
We characterized a gene cluster in a plasmid designated pPI-1 of Staphylococcus warneri ISK-1 encoding the biosynthesis of and immunity to the lacticin-481 type lantibiotic, nukacin ISK-1. The DNA sequence suggested that the nukacin ISK-1 gene cluster consists of at least six genes, nukA (a structural gene), -M, -T, -F, -E, -G, and two open reading frames, ORF1 and ORF7. NukM and NukT were predicted to be involved in post-translational modification and secretion of nukacin ISK-1 respectively. NukF, -E, and -G were predicted to form a membrane complex which contributes to self-protection from nukacin ISK-1. Transcriptional analyses revealed that nukM through ORF7 comprises an operon, and that ORF1 is transcribed independently from downstream of nukA. The transcriptional levels of the nukA and nukM genes were enhanced by osmotic stress. The expression level of the nukA transcript was scarcely enhanced by nukacin ISK-1, suggesting that expression is not under the control of the autoregulatory circuit.
Using a simple semi-synthetic competence and sporulation medium (CSM), we found evidence that Bacillus subtilis cells transformed in the competence phase can sporulate, indicating that genetic information acquired during the competence phase is inherited by the next generation after germination of the transformed spores. Moreover, the results from mixed cell culture experiments suggest that spontaneous genetic transformation can occur between competent cells and DNA released from lysed cells in the natural environment. We also found evidence that the spontaneous transformation system can be used for genetic mapping in B. subtilis.
A cDNA fragment encoding the cysteine protease inhibitor, cystatin, was cloned from pineapple (Ananas comosus) stem. This clone was constructed in a fusion vector and was easily over-expressed in Escherichia coli; satisfactory over-expression of non-fusion cystatin was achieved after an additional start codon was inserted prior to its coding sequence. Both recombinant cystatins were predominately found in the soluble fraction of the cell extract, and were demonstrated to be functionally active in a reverse zymographic assay. The fusion and non-fusion cystatins were separately purified to homogeneity via a His-tag or papain-coupling affinity column. Effective inhibitory activity against papain was detected with both the fusion and non-fusion cystatins with comparable Ki values of 1.18×10−10 M and 9.53×10−11 M, respectively. The recombinant cystatins were found to be thermally stable up to 60 °C. Inhibition of the endogenous protease activity in minced fish muscle revealed that the recombinant pineapple cystatins might be an adequate stabilizer to prevent protein degradation during industrial food processing.
Using Northern blot analysis, the expression of the Japanese chestnut (Castanea crenata Sieb. et Zucc.) agglutinin (CCA) gene was compared with that of its seed storage protein (SSP) gene. After cDNA cloning of SSP, the expression profile of SSP mRNA and CCA mRNA were compared. SSP mRNA was seed-specific, while CCA mRNA was expressed in the stems and flowers (both male and female) as well as in the seeds. Whereas extracts from all organs observed using Western blot analysis exhibited positive signals, in seeds, large expressions of SSP mRNA were restricted to the late maturation and harvest stages. Levels were maintained during the dormant period. No expression was observed during the germination stage. In contrast, CCA mRNA expression was maintained at a high level during development, was at a relatively low level during dormancy, and showed subsequent high expression during germination. These results suggest that one of the physiological roles of CCA is to act as a vegetative storage protein. But since protein expression did not coincide with that of mRNA, the expression of CCA may be regulated both at the transcription and the translation levels.
A unique variant of glutathione independent formaldehyde dehydrogenase of Pseudomonas putida was obtained by random mutagenesis using the PCR-reaction. This YM042 mutant, S318G, was a cold-adapted formaldehyde dehyrogenase. The activity at 29 °C of the variant was 1.7-fold higher than that of the wild type. The Km values of the mutant at 37 °C were 0.40 mM for NAD+ and 2.5 mM for formaldehyde, while those of the wild-type were 0.18 mM for NAD+ and 2.1 mM for formaldehyde. The catalytic efficiency for formaldehyde was about 1.5-fold greater in the mutant than in the wild-type enzyme. The optimum pHs and temperatures of the mutant and the wild-type enzyme were 7.5, and 8.0 and 37 °C, and 47 °C, respectively. The thermal stability of the mutant was lower than that of the wild type.
Tonoplast intrinsic proteins (TIPs) belong to an aquaporin family of proteins that function as water-transport channels. In this study, we isolated and characterized three novel rice cDNAs for OsTIP1, OsTIP2, and OsTIP3 that are homologous to rice γ-TIP cDNA. Northern blot hybridization analyses revealed that rice γ-TIP was expressed in all plant organs. OsTIP1 was expressed in mature seed embryos and during early seed germination. OsTIP2 was expressed exclusively in roots. OsTIP3 was specifically expressed in seeds. These results suggest that the OsTIP1, OsTIP2, and OsTIP3 genes encode discrete, functionally specialized TIPs. Immunocytochemical analysis in rice endosperm cells revealed that rice γ-TIP was localized only on the protein body type II (PB-II) membranes, whereas OsTIP3 was localized on the PB-II and the aleurone grain membranes. Although both the PB-II and the aleurone grain are derived from vacuoles, these results suggest that they may be derived from different types of vacuoles.
The ribonuclease MC1 (RNase MC1) from the seeds of the bitter gourd belongs to the RNase T2 family. We evaluated the contribution of 11 amino acids conserved in the RNase T2 family to protein folding of RNase MC1. Thermal unfolding experiments showed that substitution of Tyr101, Phe102, Ala105, and Phe190 resulted in a significant decrease in themostability; the Tm values were 47–58 °C compared to that for the wild type (64 °C). Mutations of Pro125, Gly127, Gly144, and Val165 caused a moderate decrease in thermostability (Tm: 60–62 °C). In contrast, mutations of Asp107 and Gly173 did little effect on thermostability. The contribution of Tyr101, Phe102, Pro125, and Gly127 to protein stability was further corroborated by means of Gdn–HCl unfolding and protease digestions. Taken together, it appeared that Tyr101, Phe102, Ala105, Pro125, Gly127, Gly144, Leu162, Val165, and Phe190 conserved in the RNase T2 family play an important role in the stability of the proteins.
Histidine-to-aspartate (His→Asp) phosphorelay (or two-component) systems are very common signal transduction mechanisms that are implicated in a wide variety of cellular responses to environmental stimuli in both prokaryotes and eukaryotes. Determination of the entire genomic sequence of Escherichia coli revealed that this gram-negative bacterium has 29 His-kinases and 32 response regulators. Of the 29 His-kinases, 23 have already been experimentally characterized at least to some extent in terms of their physiological functions. No physiological stimulus has yet been identified for each of the remaining 6 His-kinases (BasS, CreC, RstB, YfhK, YehU, and YpdA). Here we characterized the BasS–BasR two-component system with reference to its physiological function, taking genetic approaches together with genome-wide transcriptome profiling. First we showed that the hypothetical yfbE operon that appears to be implicated in the modification of lipopolysaccharides is regulated at the level of transcription in response to external iron, and then we showed that the BasS–BasR system is essential for this iron-dependent induction of yfbE. Another PhoQ–PhoP two-component system was also implicated in the full induction of yfbE in response to iron, but it was not essential. To gain more insight into the BasS–BasR system, we conducted genome-wide transcriptome analysis by microarray, finding that many of the uncovered putative iron-induced and BasS–BasR-dependent genes are somehow associated with acidic and/or anaerobic growth conditions. In this respect, it was found that mutant cells defective in the BasS–BasR system were sensitive to mild-acid growth conditions in the presence of a relatively high concentration of iron. These results are discussed with regard to a comprehensive picture of the His→Asp phosphorelay signaling network in E. coli.
We have developed the hyperprocessing technique to evaluate the stability of the cloverleaf shape of pre-transfer RNA (pre-tRNA). Application of this strategy to hyperprocessible human tyrosine pre-tRNA indicated that the natural intron sequence did not contribute to stabilization of the cloverleaf shape of this pre-tRNA, while the artificial intron with elongated anticodon-stem completely inhibited hyperprocessing of it. Our data suggested that the contemporary intron sequence may be a vestige of the ancient pre-biotic world, but not has been retained as a temporal stabilizer of the pre-tRNA before the base modifications.
The functional significance of amino acid residues Lys-265, Asp-270, Lys-277, Asp-288, Asp-347, Glu-349, and Arg-351 of Bacillus kaustophilus leucine aminopeptidase was explored by site-directed mutagenesis. Variants with an apparent molecular mass of approximately 54 kDa were overexpressed in Escherichia coli and purified to homogeneity by nickel-chelate chromatography. The purified mutant enzymes had no LAP activity, implying that these residues are important for the catalytic reaction of the enzyme.
The catechol 2,3-dioxygenase (C23O) gene in naphthalene catabolic plasmid pND6-1 of Pseudomonas sp. ND6 was cloned and sequenced. The C23O gene was consisted of 924 nucleotides and encoded a polypeptide of molecular weight 36 kDa containing 307 amino acid residues. The C23O of Pseudomonas sp. ND6 exhibited 93% and 89% identities in amino acid sequence with C23Os encoded by naphthalene catabolic plasmid NAH7 from Pseudomonas putida G7 and the chromosome of Pseudomonas stutzeri AN10 respectively. The Pseudomonas sp. ND6 C23O gene was overexpressed in Escherichia coli DH 5α using the lac promoter of pUC18, and its gene product was purified by DEAE-Sephacel and Phenyl-Sepharose CL-4B chromatography. The enzymology experiments indicated that the specific activity and thermostability of C23O from Pseudomonas sp. ND6 were better than those of C23O from Pseudomonas putida G7.
We isolated a cDNA encoding mitogen-activated protein kinase kinase kinase alpha, designated LjM3Kalpha, from Lotus japonicus, a model legume. The gene was expressed constitutively in roots, root nodules, and shoots. We also identified a novel nodulin gene, LjNUF, that shows specific expression in nodules. LjNUF resembles the C-terminal half of a hypothetical protein (pir//D85436), the N-terminal half of which is similar to a portion of mitogen-activated protein kinase kinase kinase gamma. Although LjNUF was predicted to be a secreted protein, its function remains to be clarified.
To identify the interaction proteins for the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunit glutamate receptor-interacting protein 1 (GRIP1), GRIP1 interactions with microtubule-associated protein (MAP)-1B light chain (LC) were investigated. GRIP1 interacts with MAP-1A and MAP-1B in the yeast two-hybrid assay, as is indicated also by glutathione S-transferase (GST) pull-down and coimmunoprecipitation with MAP-1B LC antibody in brain fractions. These results suggest a novel mechanism for localizing AMPA receptors to synaptic sites.
Salt-activation of thermolysin was examined using a positively charged fluorescent substrate, (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH2 [MOCAc-PLGL(Dpa)AR]. Thermolysin activity increased in a biphasic exponential fashion and was 40 times higher in the presence of 4 M NaCl than in its absence. The degree of activation at x M NaCl was expressed as 4.7x when [NaCl]o<0.5 M and 2.3x when [NaCl]o>0.5 M respectively.
Rice anthers contain high concentrations of gibberellins A4 and A7. To understand their physiological roles, we examined the site of their biosynthesis by analyzing the expression pattern of a gene (OsCPS) encoding coparyl diphosphate synthase in developing rice flowers. Expression was apparent in the anthers 1–2 days before flowering, and CPS mRNA accumulated in the maturing pollen.
The effects of (−)-epigallocatechin gallate (EGCG) on the contraction of floating collagen gel by fibroblasts were investigated. EGCG inhibited collagen gel contraction dose-dependently. On the basis of the fact that platelet-derived growth factor (PDGF) is one of the serum components with stimulatory activity in collagen gel contraction, we examined the possibility that interaction between EGCG and PDGF may be involved in this inhibition mechanism. We confirmed this by recombinant PDGF-BB in the present system and we found that EGCG inhibited PDGF-stimulated collagen gel contraction. The results of affinity chromatography indicated that PDGF was bound by EGCG immobilized on agarose gel as detected by enzyme-linked immunoassay and Western blotting. These findings suggest that binding of EGCG to PDGF is at least partly involved in the mechanism of inhibition of collagen gel contraction by EGCG.
Silkworm larvae at the 5th instar were injected with lipopolysaccharide from Escherichia coli and inducible polypeptides were examined within a pI range of 3–10 and a size range of 14–97 kDa by proteomics, including peptide mass fingerprinting. No polypeptides were induced in the midgut. FB1 and H1–4 polypeptides were significantly induced in fat body and hemolymph, respectively. FB1 and H1 were estimated to be antitrypsin and serpin-2 proteinase inhibitors respectively. H2 and H3 were novel polypeptides. H4 was estimated to be attacin antibacterial polypeptide with high coverage of sequence. The amounts of all the induced polypeptides decreased at 48 h after the injection.
Chard (Beta vulgaris L. var cicla) is one of the medicinal herbs used by diabetics in Turkey. It has been reported to reduce blood glucose. We have investigated the effect of chard extracts on the liver by biochemical and morphological investigation. The plant extract was administered by the gavage technique to rats at a dose of 2 g/kg every d for 28 d, 14 d after experimental animals were made diabetic. In the diabetic group, some degenerative changes were observed by light and electron microscope examination, but degenerative changes decreased or were not observed in the diabetic group given chard. In the diabetic group, blood glucose levels, serum alanine, aspartate transaminase, alkaline phosphatase activities, total lipids, sialic and uric acid levels, liver lipid peroxidation (LPO), and nonenzymatic glycosylation (NEG) levels increased, while blood glutathione, body weight, and liver glutathione (GSH) levels decreased. The diabetic group given chard, serum alanine, aspartate transaminase, alkaline phosphatase activities, total lipid level, sialic and uric acid levels, blood glucose levels, and liver LPO and NEG levels decreased, but the other values increased. As a result of all the morphological and biochemical findings obtained, it was concluded that the extract of this plant has a protective effect on the liver in diabetes mellitus.
This study was conducted to identify the sourness-suppressing peptides in cooked pork and to clarify the mechanism of sour taste suppression by the peptides. An extract prepared from pork loins vacuum-cooked at 60 °C for 6 hours after conditioning at 4 °C for 20 days was separated into three fractions: under MW 500 (Fraction I), MW 500–1,000 (Fraction II), and over MW 1,000 (Fraction III). The Fraction I content was largest. As judged by sensory evaluation, the addition of Fraction II was capable of suppressing stronger sourness than the other fractions. Fraction II also enhanced umami and saltiness. Three peptides (APPPPAEVHEVV, APPPPAEVHEVVE, and APPPPAEVHEVHEEVH) in Fraction II increased greatly during conditioning. A common peptide, APPPPAEVHEV, in the amino acid sequences of the three peptides suppressed the sour taste. The mechanism of sourness suppression by the peptide was concluded to comprise inhibition of the binding of sour taste substances to the membranes of the tongue.
Citrus reticulata Blanco (ponkan) cold-pressed oil and its oxygenated fraction were studied by analytical (GC and GC/MS) and sensory analyses. The monoterpene group was predominant, accounting for more than 89.6% (w/w), of which limonene was the most abundant (80.3%). Among the oxygenated compounds, octanal and decanal were the major ones among 12 aldehydes accounting for >1.5%; six alcohols were identified with a total concentration of >0.7%, while oxides, ketones and esters did not quantitatively or qualitatively contribute to the oil. Sniffing the ponkan cold-pressed oil and its oxygenated fraction demonstrated that octanal and decanal were the characteristic odor components of ponkan. Reconstruction of the ponkan aroma model and its sensory evaluation by a hedonic test were performed, showing that, in addition to octanal and decanal which played important roles, (R)-(+)-limonene contributed to the aroma model as a background component, making the aroma model very similar to that of the original.
The anti-allergic activity of a tomato extract was studied by using an in vitro histamine-release assay. The tomato skin extract exerted the strongest inhibition of histamine release. Chlorogenic acid, rutin and naringenin were identified in the 60% ethanol extract of tomato skin. However, the extract contained an unknown compound which strongly inhibited histamine release. This active compound in tomato skin was identified as naringenin chalcone (trans-2′4′6′4-tetrahydroxychalcone). Naringenin chalcone inhibited histamine release with an IC50 value of 68 μg/ml. The anti-allergic activity of the tomato skin extract was next investigated by the in vivo mouse ear-swelling response. We found that naringenin chalcone showed the strongest inhibitory effect of the polyphenols of the tomato skin extract. These results indicate that a tomato skin extract could inhibit allergic reactions.
Psyllium husk, a highly viscous fiber, has beneficial effects for health, but has been reported to inhibit absorption of calcium. The present study found the effects of fiber viscosity on calcium, magnesium, and zinc absorption with partially degraded psyllium preparations to be one fifth viscosity (LD-HP) and one seventieth viscosity (HD-HP) using normal and ovariectomized rats. Magnesium absorption was reduced with ingestion of intact psyllium (50 g/kg diet) for 4 weeks but this reduced absorption was increased with lower viscous psyllium preparations. Moreover, the absorption in the HD-HP group was higher than in the control group (100 g cellulose/kg diet) in ovariectomized rats. Changes in calcium and zinc absorption were similar to those in magnesium absorption. Cecal pH was reduced only in rats fed HD-HP in both normal and ovariectomized rats. These results indicate that reduction of psyllium viscosity tends to counter inhibitory effects on mineral absorption.
The interaction of a tea catechin, epigallocatechin gallate (EGCg), with the model membrane of dimyristoylphosphatidylcholine (DMPC) was studied by solid-state 31P and 2H NMR. The 31P chemical shift anisotropy of the DMPC phosphate group decreased on addition of EGCg. The 2H NMR spectrum of [4-2H]EGCg, which is deuterated at the 4-position, in the DMPC liposomes gave deuterium nuclei with much smaller quadrupole splittings than those in the solid phase. These 31P and 2H NMR observations provide direct experimental evidence that the EGCg molecule interacts with the lipid bilayers.
The effect of CPP-III, a commercially available casein phosphopeptide, on the mRNA expression of cytokines in Caco-2 cells was investigated. CPP-III enhanced the mRNAs of interleukin (IL)-6 and tumor necrosis factor-α while IL-1β was not affected. The mRNA expression of IL-6 was stronger in the presence of both CPP-III and bacterial components such as peptidoglycan from Lactobacillus acidophilus and lipopolysaccharide from Salmonella typhimurium. These results suggest that CPP-III influences the expression of cytokines in intestinal epithelial cells.
In order to test the estrogenic activity of sterol oxidation products from cholesterol and phytosterols, an estrogen-dependent gene expression assay was performed in estrogen receptor α-stably transformed HeLa cells. The ranking of the estrogenic potency of these compounds was different: 17β-estradiol >> genistein >> β-epoxycholesterol = daidzein = cholestanetriol = 22(R)-hydroxycholesterol = 20(S)-hydroxycholesterol = sitostanetriol > campestanetriol = β-epoxysitosterol = 7β-hydroxycholesterol. These compounds were not estrogenic in estrogen receptor-negative HeLa cells.
Microorganisms were screened for their ability to release cadmium from scallop hepatopancreas, which is the main residue after removing of the edible parts of scallop. The isolated strain, 23-0-11, identified as Arthrobacter nicotinovorans, secreted a protease which released cadmium from scallop hepatopancreas into the liquid medium. The molecular mass of the enzyme was estimated to be 27 kDa. The sequence of the 15 N-terminal amino acids of the protease showed no close similarity with any other protein. Compared with a commercial enzyme, the purified protease had greater ability to release cadmium. The enzyme activity was greatest at 50 °C and pH 7.0, and was enhanced in the presence of Ca2+, Mg2+ and Mn2+, while being strongly inhibited by Co2+. The inhibition profile by the serine protease inhibitor, phenylmethylsulphonyl fluoride (PMSF), confirmed that the protease belonged to the serine protease family.
Flavin reductase is essential for the oxygenases involved in microbial dibenzothiophene (DBT) desulfurization. An enzyme of the thermophilic strain, Bacillus sp. DSM411, was selected to couple with DBT monooxygenase (DszC) from Rhodococcus erythropolis D-1. The flavin reductase was purified to homogeneity from Bacillus sp. DSM411, and the native enzyme was a monomer of Mr 16 kDa. Although the best substrates were flavin mononucleotide and NADH, the enzyme also used other flavin compounds and acted slightly on nitroaromatic compounds and NADPH. The purified enzyme coupled with DszC and had a ferric reductase activity. Among the flavin reductases so far characterized, the present enzyme is the most thermophilic and thermostable. The gene coded for a protein of 155 amino acids with a calculated mass of 17,325 Da. The enzyme was overproduced in Escherichia coli, and the specific activity in the crude extracts was about 440-fold higher than that of the wild-type strain, Bacillus sp. DSM411.
Bacillus subtilis YkvV protein, an extracellular thioredoxin superfamily protein, was successfully expressed both in Brevibacillus choshinensis culture medium using an efficient promoter and the secretion signal of its surface layer protein, and in Escherichia coli cytoplasm with the amino-terminal His-tag (His-YkvV). His-YkvV was purified to homogeneity by Ni-NTA column. Both secreted YkvV and purified His-YkvV exhibited thiol-disulfide oxidoreductase activity.