Protein quality control (QC) in the endoplasmic reticulum (ER) comprises many aspects, including folding and transport of nascent proteins and degradation of misfolded proteins. Recent studies have revealed that high-mannose-type glycans play pivotal roles in the QC process. To gain knowledge of the molecular basis of this process with well-defined homogeneous compounds, we achieved a convergent synthesis of high-mannose-type glycans and their functionalized derivatives. A major part of our study focused on analyses of UDP-Glc: glycoprotein glucosyltransferase (UGGT) and ER glucosidase II, which play crucial roles in glycoprotein QC, to clarify their specificities. In addition, we established an in vitro assay system mimicking the in vivo condition, which is highly crowded due to the presence of various macromolecules.
Two new phenolic compounds, caesalpiniaphenols G–H (1 and 2), were isolated from Vietnamese Caesalpinia sappan heartwood. The chemical structures were established mainly by extensive spectroscopic studies and chemical evidence. Compounds 1 and 2 showed potent inhibitory activity against HL-60 cancer cell lines with respective IC50 values of 16.7 and 22.5 µg/mL. Treating HL-60 cells with various concentrations of 1 resulted in growth inhibition and the induction of apoptosis.
Maillard reaction inhibitors could be useful therapeutics for diabetes and other age-related diseases. We isolated for the first time 4-O-demethylsilvaticol (1) and (−)-mitorubrin (2) as Maillard reaction inhibitors from Paecilomyces sp. 3193B. Among the isolated inhibitors, 2 showed most potent inhibitory effect by an SDS–PAGE assay on cross-linked protein formation and by a fluorescent assay on AGE formation.
1'-Acetoxychavicol acetate (ACA), extracted from rhizomes of tropical ginger, possesses antitumor properties against a wide variety of malignancies. MicroRNAs have been found to act as oncogenes and as tumor suppressor genes in the development of cancer. The purpose of this study was to investigate the miRNA involved in the molecular mechanisms of ACA action on tumor inhibition. It was found that ACA significantly inhibited the growth of human head and neck squamous cell carcinoma cell line HN4 and induced cell apoptosis. Further studies indicated that ACA downregulated the expression of miR-23a in HN4 cells. Transfection with anti-miR-23a inhibited the proliferation of HN4 cells and induced cell apoptosis. In addition, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) was confirmed to be the target of miR-23a. Taken together, our findings suggest that ACA might have anticancer effects against human head and neck cancer through downregulation of miR-23a, which can repress tumor suppressor PTEN.
The biological activity of Mastixia arborea (MA) relates to inflammation, but the underlying mechanisms are largely unknown. We confirmed the anti-inflammatory effects of a methanol extract of MA extract on lipopolysaccharide (LPS)-stimulated RAW264.7 mouse macrophage cells and carrageenan-induced mice paw edema. The MA extract significantly inhibited nitric oxide (NO), prostaglandin E2 (PGE2), interleukin-1β (IL-1β), and IL-6 production in LPS-stimulated RAW264.7 cells. In vitro expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) was suppressed by the extract. The extract attenuated acute inflammatory responses in carrageenan-induced mice paw edema. A mechanism study indicated that translocation of the NF-κB (p65) subunit into the nucleus and phosphorylation of ERK and JNK were inhibited by the extract. These results indicate that the extract is an effective suppressor of the inflammatory response, blocking the phosphorylation of ERK and JNK and the translocation of NF-κB in macrophages, thereby producing an anti-inflammatory effect in vivo.
Tyrosinase exists in three forms in the catalytic cycle depending on the oxidation state of the copper: met- (Em), oxy- (Eox), and deoxy- (Ed). When O-quinones, products of the enzymatic reaction, evolve chemically to generate an O-diphenol in the reaction medium, the enzyme acts on a monophenol with O-diphenol as reductant, converting Em to Ed. The binding of Ed to molecular oxygen gives Eox, which is active on monophenols, but when the O-quinone product does not generate O-diphenol through chemical evolution, the monophenol does not act as an enzyme substrate. The fact that Eox can be formed from Em with hydrogen peroxide can be used to help identify whether a monophenol is a substrate of tyrosinase. The results obtained in this study confirm that compounds previously described as inhibitors of the enzyme are true substrates of it.
Hypertrophic scar (HS) is a dermal fibroproliferative disorder characterized by excessive deposition of extracellular matrix. Here, to investigate the regulative effects of resveratrol, a natural antioxidant compound, on fibroblasts from human skin HS tissue, a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate the inhibitory effect of resveratrol on cells. Cellcycle progression and apoptosis were measured by flow cytometry and Hoechst 33258 staining respectively. The hydroxyproline content and mRNA expression levels of type I and III procollagen were measured separately by ELISA and reverse transcription-polymerase chain reaction (RT-PCR). The results indicated that resveratrol significantly inhibited cell growth, arresting the cell cycle at the G1 phase and inducing apoptosis in the fibroblasts, decreasing hydroxyproline (or collagen) levels, and downregulating the expression levels of type I and III procollagen mRNA. Taken together, these data indicate that resveratrol-mediated reduction of collagen in fibroblasts is at least partially effected by causing inhibitory cell growth, cellcycle arrest, and apoptosis, and they suggest that resveratrol is a potential agent for HS treatment.
The IGF-IR/PI3K/Akt signaling pathway inhibited GSK3-β activity by phosphorylation and this promoted β-catenin nuclear localization. Our previous study indicated that β-catenin mRNA level was significantly higher in tumor areas than in non-tumor ones, especially in late pathologic stage tumors. However, β-catenin inhibition resulted in significantly suppressed migration and invasion ability of HA22T cells. Thus, Wnt/β-catenin pathway over-activation might be involved in metastatic enhancement of apicidin-resistant HA22T cell metastasis. Apicidin-resistant (AR) HA22T cells showed higher β-catenin nuclear accumulation and significantly decreased GSK-3-β protein level, in relation to parental cells. Results also indicated that AR cells increased abundantly in Tbx3, a downstream target of Wnt/β-catenin that it is implicated in liver cancer. AR cells also inhibited the MEK/ERK/PEA3 pathway which promoted MMP-2 activation. But, apicidin-resistant effect was totally reversed by LY294002 and AG1024. In conclusion, Apicidin-R HA22T cells activated the Wnt/β-catenin pathway and induced, MMP-2 expression via IGF-IR/PI3K/Akt signaling further enhancing cell the metastatic effects.
Metabolic syndrome and related disorders are increasingly prevalent in contemporary society, and thus pose the need for potent agents to control lipid accumulation in the body. This study indicates that Caenorhabditis elegans was effective in screening for potent lipid metabolism modulators with berberine as a model compound. Among the various isoquinoline alkaloids tested, sanguinarine, a benzophenanthridine alkaloid, was found to be the most potent. Sanguinarine, like berberine, reduced lipid accumulation through AMP-activated protein kinase activation. Analysis of AMPK (aak-1 and aak-2) RNAi worms revealed that effects were aak-2-dependent. Characterization of worms with knockdown nhr-49, a hormone nuclear receptor gene that functions as a key regulator of fat consumption, showed that both alkaloids were effective even in these markedly lipid-accumulating nhr-49 RNAi worms, suggesting that they predominantly affect lipid synthesis, rather than fatty acid β-oxidation. The versatility of C. elegans for the purpose of lipid-modulating chemical screening and characterization of the underlying mechanisms is discussed.
Linalool has two enantiomers, (R)-linalool and (S)-linalool. Both are known to possess several biological activities in stressed animals. Our previous work revealed that inhalation of (R)-linalool altered hypothalamic gene expression in rats under stress. In the present study, we monitored hypothalamic gene expression in restrained rats with and without (S)-linalool inhalation by DNA microarray. The entire gene expression profile showed that inhalation of (S)-linalool significantly changed the expression levels of 316 hypothalamic genes in the restrained rats. The differentially expressed genes (e.g., App, Avp, Igf2, Igfbp2, Sst and Syt5) were found to relate to cell-to-cell signaling and nervous system development. These results indicate that (S)-linalool influences hypothalamic gene expression in restrained rats, and that inhalation of (S)-linalool under the stressed condition has some effects on stress-related biological responses.
To elucidate the scavenging systems of sugar- and lipid-derived reactive carbonyls (RCs) in the cyanobacterium Synechocystis sp. PCC 6803 (S. 6803), we selected proteins from S. 6803 based on amino-acid (AA) sequence similarities with proteins from Arabidopsis thaliana, and characterized the properties of the GST-fusion proteins expressed. Slr0942 catalyzed the aldo-keto reductase (AKR) reaction scavenging mainly sugar-derived RCs, methylglyoxal (MG). Slr1192 is the medium-chain dehydrogenase/redutase (MDR). It catalyzed the AKR reaction scavenging several lipid-derived RCs, acrolein, propionaldehyde, and crotonaldehyde. Slr0315 is a short-chain dehydrogenase/redutase (SDR), and it catalyzed only the reduction of MG in the AKR reaction. Slr0381 catalyzed the conversion of hemithioacetal to S-lactoylglutahione (SLG) in the glyoxalase (GLX) 1 reaction. Sll1019 catalyzed the conversion of SLG to glutathione and lactate in the GLX2 reaction. GLX1 and GLX2 compose the glyoxalase system, which scavenges MG. These enzymes contribute to scavenging sugar- and lipid-derived RCs as scavenging systems.
Plant elongation growth on a day-to-day basis is enhanced under specific photoperiod and temperature conditions. Circadian clock is involved in the temperature adaptive photoperiodic control of plant architecture, including hypocotyl elongation in Arabidopsis thaliana. In this regulation, phytochrome interacting transcriptional factors, PIF4 and PIF5, are activated at the end of night under short photoperiod or high temperature conditions, due to the coincidence between internal (circadian rhythm of the transcripts) and external (length of dark period) time cues. It is previously found that biosynthesis or metabolism of phytohormones including auxin, and their signal transduction-related genes are downstream targets of circadian clock and PIF4/PIF5 mediated external coincidence mechanism. Brassinosteroid and gibberellic acid played a positive role in the hypocotyl elongation of seedlings under light and dark cycle conditions. On the other hand, cytokinin and jasmonic acid played an opposite role. In this study, diurnal expresson profile of a gene encoding a sulfotransferase family protein that is involved in the jasmonic acid metabolism, ST2A, was examined. It was found that transcription of ST2A is induced at the end of night under LD/22 °C and SD/28 °C conditions according to the external coincidence mechanism. The results of this study support the idea that the circadian clock orchestrates a variety of hormone-signaling pathways to regulate the photoperiod and temperature-dependent morphogenesis in A. thaliana.
Saccharomycescerevisiae strains engineered previously to produce proteins with mammalian high mannose structures showed severe growth defects and decreased protein productivity. In strain YAB101, derived from one of these strains by a mutagenesis technique based on the disparity theory of evolution, these undesirable phenotypes were alleviated. Here we describe further engineering of YAB101 with the aim of synthesizing heterologous glycoproteins with Man5GlcNAc2, an intermediate for the mammalian hybrid and complex type oligosaccharides. About 60% conversion of Man8GlcNAc2 to Man5GlcNAc2 was observed after integration of Aspergillussaitoi α-1,2-mannosidase fused to the transmembrane domain of S. cerevisiae Och1. To obtain a higher yield of the target protein, a protease-deficient version of this strain was generated by disruption of PEP4 and PRB1, resulting in YAB101-4. Inactivation of these vacuolar proteases enhanced the secretion of human interferon-β by approximately 10-fold.
Desferrioxamines E (1), D2 (2), X1 (3), and X2 (4), four macrocyclic N-hydroxy-N-succinyl diamine-based siderophores, were produced efficiently by heterologous expression of a fusion biosynthetic gene cluster. This expression system consisted of three genes (mbsA-C) from marine metagenomic DNA and one gene (dfoCC) from the terrestrial bacterium Erwinia amylovora. The first three genes are functional in the production of the common monomers N-hydroxy-N-succinyl cadaverine (5, HSC) and N-hydroxy-N-succinyl putrescine (6, HSP), whereas dfoCC catalyzes the oligomerization and the macrocyclization reactions of compounds 5 and 6 to form compounds 1–4. This fusion gene cluster system provides a convenient expression platform for various biosynthetic genes of HSC-HSP based siderophores by simply switching the fourth gene by the cassette process.
Escherichia coli RNase G is involved in the degradation of several mRNAs, including adhE and eno, which encode alcohol dehydrogenase and enolase respectively. Previous research indicates that the 5' untranslated region (5'-UTR) of adhE mRNA gives RNase G-dependency to lacZ mRNA when tagged at the 5'-end, but it has not been elucidated yet how RNase G recognizes adhE mRNA. Primer extension analysis revealed that RNase G cleaved a phosphodiester bond between −19A and −18C in the 5'-UTR (the A of the start codon was defined as +1). Site-directed mutagenesis indicated that RNase G did not recognize the nucleotides at −19 and −18. Random deletion analysis indicated that the sequence from −145 to −125 was required for RNase G-dependent degradation. Secondary structure prediction and further site-directed deletion suggested that the stem-loop structure, with a bubble in the stem, is required for RNaseG-dependent degradation of adhE mRNA.
We describe a transient dual-luciferase assay combined with a glucocorticoid-inducible system for rice protoplasts. Luciferase genes were efficiently induced by adding 0.1 µM of dexamethasone to the protoplast suspension, the activity of the luciferases reaching a maximum 6 h after induction. This assay system is applicable to studying the translation efficiency of rice by using the luciferase gene harboring tandem copies of an interesting codon at the 5' end.
We measured the antioxidant contents and antioxidative activities in eight Allium fistulosum–shallot monosomic addition lines (MAL; FF+1A–FF+8A). The high antioxidative activity lines (FF+2A and FF+6A) showed high polyphenol accumulation. These additional chromosomes (2A and 6A) would therefore have anonymous genes related to the upregulation of polyphenol production, the antioxidative activities consequently being increased in these MALs.
Lily pollen was developed to express HBsAg by Agrobacterium-mediated transformation. A double prime-boost strategy combining parenteral and oral HBsAg boosters was found to increase antibody titer levels 10-fold, as compared to a single process of priming and boosting. Lily pollen may prove a tool for oral vaccine development.
A thermostable acetylxylan esterase gene, TTE0866, which catalyzes the deacetylation of cellulose acetate, was cloned from the genome of Caldanaerobacter subterraneus subsp. tengcongensis. The pH and temperature optima were 8.0 and 60 °C. The esterase was inhibited by phenylmethylsulfonyl fluoride. A mixture of the esterase and cellulolytic enzymes efficiently degraded insoluble cellulose acetate with a higher degree of substitution.
We found that brain-derived neurotrophic factor (BDNF)-induced phosphorylation of mitogen-activated protein kinase (MAPK) and Akt in cerebellar granule neurons was specifically potentiated by LPC. LPC also augmented the BDNF-induced phosphorylation of TrkB, the receptor for BDNF. In TrkB-transfected CHO-K1 cells, LPC potentiated BDNF-induced MAPK phosphorylation. These results suggest that LPC plays a role in BDNF-TrkB signaling by regulating the activation of TrkB.
Peach trees bear either white- or yellow-flesh fruit. We found that Japanese peach cultivars have two types of mutation in a carotenoid catabolic gene, CCD4: the insertion of a retrotransposon, and a frame shift in the microsatellite sequences of the first exon. CCD4 in yellow-flesh peaches was disrupted by these mutations.
(−)-Tulipalin B and (+)-6-tuliposide B were confirmed to inhibit MurA in vitro. However, contrary to fosfomycin, these compounds showed potent inhibitory activities against MurA overexpressing Escherichia coli, especially in the presence of UDP-GlcNAc. These observations suggest that these compounds induced bacterial cell death not through a MurA malfunction, but in such a MurA-mediated indirect manner as the inhibition of other Mur enzymes.
The levels of food allergens in gamma-irradiated soybean (0, 2.5, 5, 7.5, 10, 20, and 30 kGy) were investigated by immunoblotting and ELISA, using allergen-specific antibodies and patient serum. After 3 months of storage, Coomassie brilliant blue (CBB) staining indicated similar total protein profiles among the treatments, but that some proteins were degraded by irradiation at high doses. Immunoblotting with specific antibodies for major soybean allergens (β-conglycinin, Gly m Bd 30 K, soybean trypsin inhibitor, and Gly m 4) resulted in apparent band profiles and intensities that were not significantly changed by irradiation. Competitive inhibition ELISA analyses suggested that there were no significant changes in the allergen contents, except for a decrease in the soybean trypsin inhibitor. The patient IgE binding allergenic protein patterns were not changed by irradiation up to 30 kGy. ELISA using patient serum also revealed that the IgE reactivity to the irradiated soybean extract did not increase from the level of the control, but that the reactivity to some patient serum IgE was significantly decreased by irradiation.
Cooked grains of ae rice cultivars are too hard and non-sticky due to the presence of long-chain amylopectin, and ae rice cultivars are therefore called ``super-hard rice'' and cannot be used as table rice. However, they are promising in terms of their bio-functionality such as preventing diabetes. Miso (soybean paste) is a yeast-fermented food, made from steamed soybeans, salt, and inoculated cereals known as koji, made from rice, barley, or soybeans. We investigated the effects of soaking ae mutant rice cultivars in a miso suspension. Their chemical components, physical properties, and enzyme activities were measured under different conditions (milled rice before or after soaking in a 5% barley-koji miso suspension). Rice grains cooked after soaking in the miso suspension were less hard and more sticky than those cooked after soaking in water. Rice grains cooked after soaking in a 5% barley-koji miso suspension maintained high amounts of resistant starch and dietary fiber, and were fortified with polyphenols and isoflavones. Palatable and bio-functional ae rice could therefore be produced by cooking after soaking in a 5% barley-koji miso suspension.
We tested the peroxisome proliferator-activated receptor (PPAR)δ agonistic activity of a Garcinia mangostana pericarp extract to develop a treatment for the metabolic syndrome, and demonstrated γ-mangostin to be an active compound on the basis of a luciferase reporter gene assay. γ-Mangostin induced the expression of the uncoupling protein-3 (UCP-3) gene which is related to energy expenditure and fat metabolism in L6 cells. We showed that γ-mangostin is a dual agonist that activates both PPARδ and PPARα. γ-Mangostin also induced the expression of acyl-CoA synthase and carnitine palmitoyl-transferase 1A genes in HepG2 cells. These results suggest the potential of γ-mangostin as a preventive agent of the metabolic syndrome.
The cyanobacterium semi-filamentous multicellular strain ABRG5-3 undergoes cell lysis as a unique feature that occurs due to growth condition changes from normal cultivation with shaking to static cultivation without shaking in liquid culture (Nishizawa et al., 2010). Microscopic observation and energy dispersive X-ray spectrometer (EDX) analysis have revealed that lysis is involved in the accumulation of polyphosphate compounds and the disintegration of thylakoid membranes in cells. Static cultivation, dark or red light exposure, and temperature (22 to 42 °C) conditions were found to be effective factors for the induction of lysis. Moreover, stress induced by salts, osmotic pressure with sucrose, and the depletion of nitrogen or phosphate in cultures also induced ABRG5-3 cell lysis. Based on these results, we discuss lysis and its utilization in the biotechnology industry.
Cyberlindnera samutprakarnensis JP52T, isolated from cosmetic industrial wastes in Thailand, was found to be an efficient biosurfactant-producing yeast when cultured in a medium containing (2% (w/v) glucose and 2% (v/v) palm oil at 30 °C, 200 rpm for 7 d. The crude biosurfactant had the ability to reduce the surface tension from 55.7 to 30.9 mN/m at 25 °C with a critical micelle concentration (CMC) of 0.046%. Physicochemical analysis of the crude biosurfactant revealed that it had wide ranges of optimum pH and pH stability at 6–9 and 3–10 respectively. It was also thermostable and retained 80% activity even after heat treatment, and it tolerated NaCl at 1.0–10%. Furthermore, it effectively emulsified various vegetable oils with an E24 value of over 80%. A partially purified biosurfactant fraction was analyzed for its structure by MALDI-TOF MS and NMR. This revealed that the biosurfactant mainly contained sophorolipids in C18-(MW 574) and C16-diaceltylated (MW 662) forms.
N-Acylhomoserine lactones (AHLs) function as quorum-sensing signaling molecules in many Gram-negative bacteria. We isolated a total of 672 bacterial strains from activated sludge obtained from seven sewage treatment plants in Tochigi Prefecture, Japan, and screened for AHL-producing and degrading strains. Isolates (n=107) stimulated AHL-mediated purple pigment production in AHL reporter strains Chromobacterium violaceum CV026 and VIR07. Based on their 16S rRNA gene sequences, most of these AHL-producing isolates were assigned to the genus Aeromonas, and they were divided into six groups. Isolates (n=46) degraded N-decanoyl-L-homoserine lactone (C10-HSL) within 24 h. Based on their 16S rRNA gene sequences, the most dominant AHL-degrading isolates were assigned to the genus Acinetobacter and divided into six groups. Strains Ooi24, Omo91, and Uzu81, which showed higher C10-HSL-degrading activity, showed putative AHL-acylase activity.
Amylases from Streptomyces are useful in the production of maltooligosaccharides, but they have weak thermal stability at temperatures higher than 40 °C. In this study, α-amylase (SAV5981 gene of Streptomyces avermitilis) was expressed from Streptomyces lividans 1326 and purified by ammonium sulfate fractionation followed by anionic chromatography (Q-HP sepharose). The properties of the purified SAV5981 amylase were determined by the starch-iodine method. The effect of metal ions on amylase activity was investigated. The optimal temperature shifted from 25 to 50 °C with the addition of the Ca2+ ion. The thermal stability of SAV5981 was also dramatically enhanced by the addition of 10 mM CaCl2. Improvement of the thermal stability of SAV5981 was examined by CD spectra in the presence and the absence of the Ca2+ ion. Thin-layer chromatography (TLC) analysis and HPLC analysis of starch degradation revealed that SAV5981 mainly produced maltose and maltotriose, not glucose. The maltoorigosaccharide-producing amylase examined in this study has the potential in the industrial application of oligosaccharide production.
The Antarctic basidiomycetous yeast Mrakia blollopis SK-4 fermented ethanol between pH 5.0 and pH 10.0 with optimum pH at 8.0–10.0. Knowledge of ethanol fermentability as to the genus Mrakia remains incomplete. Further experiments are required to elucidate the ethanol fermentability of genus e.g., as to optimum fermentation pH, optimum fermentation temperature, and cell viability during fermentation.
The antibacterial effects of salivary nitrate/nitrite on the growth of three Desulfovibrio species were examined. The bacteria did not grow on plates with ≥0.2 mM nitrate or ≥1.0 mM nitrite. They were also incubated in filter-sterilized saliva. D. desulfuricans was reduced on the order of >102 compared with the control solution (phosphate-buffered saline) in nine out of the 10 participants.
A gene, CmCDR1, encoding an ABC transporter of the dicarboxylic acid (DCA)-producing yeast Candida maltosa was cloned. Transcription of CmCDR1 was upregulated in a DCA-hyper-producing mutant of C. maltosa in a later phase of culture on n-dodecane, but not in its parental strain. CmCDR1 expression was significantly induced by the longer-chain DCA in this mutant.
In the fermentation industry, the traceability of microorganisms during the process is important to ensure safety and efficacy. Ethyl carbamate, a group-2A carcinogen, is produced from ethanol and urea during the storage of food/alcoholic beverages. We isolated non-urea-producing sake yeast car1 mutants carrying a discriminable molecular marker, and demonstrated, by the use of PCR assays, that these mutants are useful for traceability analysis and identification during the sake brewing process.
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