In vitro protein import of a putative amino acid transporter from Arabidopsis thaliana into chloroplasts and its suborganellar localization

We identified a gene product of At5g19500 (At5g19500p) from Arabidopsis thaliana that is homologous to EcTyrP, a tyrosine-specific transporter from Escherichia coli. Computational analyses of the amino acid sequence of At5g19500p predicted 11 transmembrane domains (TMDs) and a potential plastid targeting signal at its amino terminus. As a first step toward understanding the possible role of At5g19500p in plant cells, we attempted to determine the localization of At5g19500p by an in vitro chloroplastic import assay using At5g19500p translated in a cell-free wheat germ system (Madin et al., Proc. Natl. Acad. Sci. USA, 97, 559-564 (2000)), followed by subfractionation of the chloroplasts. At5g19500p was successfully imported into chloroplasts, and the newly transported mature form of At5g19500p was recovered from the inner envelope membrane.     

Quercetin appears in the lymph of unanesthetized rats as its phase II metabolites after administered into the stomach

Quercetin is a major flavonoid in plant foods and potentially has beneficial effects on disease prevention. The present work demonstrated that quercetin was transported into the lymph after being metabolized in the gastrointestinal mucosa of rats. Glucuronide/sulfate and methylated conjugates of quercetin appeared in the lymph, but not quercetin aglycone. The highest lymphatic concentration was found at as rapid as 30 min after administration, suggesting gastric absorption, whereas the mucosal glucuronidation activity was significantly higher in the duodenum and jejunum than in the stomach. This is the first report to show the lymphatic flavonoid transport pathway from the gastrointestinal tract.     

Scavenger receptor expressed by endothelial cells I (SREC-I) mediates the uptake of acetylated low density lipoproteins by macrophages stimulated with lipopolysaccharide

Scavenger receptor expressed by endothelial cells I (SREC-I) is a novel endocytic receptor for acetylated low density lipoprotein (LDL). Here we show that SREC-I is expressed in a wide variety of tissues, including macrophages and aortas. Lipopolysaccharide (LPS) robustly stimulated the expression of SREC-I in macrophages. In an initial attempt to clarify the role of SREC-I in the uptake of modified lipoproteins as well as in the development of atherosclerosis, we generated mice with a targeted disruption of the SREC-I gene by homologous recombination in embryonic stem cells. To exclude the overwhelming effect of the type A scavenger receptor (SR-A) on the uptake of Ac-LDL, we further generated mice lacking both SR-A and SREC-I (SR-A(-/-);SREC-I(-/-)) by cross-breeding and compared the uptake and degradation of Ac-LDL in the isolated macrophages. The contribution of SR-A and SREC-I to the overall degradation of Ac-LDL was 85 and 5%, respectively, in a non-stimulated condition. LPS increased the uptake and degradation of Ac-LDL by 1.8-fold. In this condition, the contribution of SR-A and SREC-I to the overall degradation of Ac-LDL was 90 and 6%, respectively. LPS increased the absolute contribution of SR-A and SREC-I by 1.9- and 2.3-fold, respectively. On the other hand, LPS decreased the absolute contribution of other pathways by 31%. Consistently, LPS did not increase the expression of other members of the scavenger receptor family such as CD36. In conclusion, SREC-I serves as a major endocytic receptor for Ac-LDL in LPS-stimulated macrophages lacking SR-A, suggesting that it has a key role in the development of atherosclerosis in concert with SR-A.     

Covalent circularization of exogenous RNA during incubation with a wheat embryo cell extract

Cell extracts from wheat embryos have been widely used for mRNA-directed protein production. Here, we found that a significant fraction of exogenous linear RNAs are circularized in a wheat embryo extract. The circularization was seen only in uncapped RNAs. The amount of the circular species reached around 1% of the initial RNA and increased along with an increase in the initial concentration more than proportionally. The circular RNAs were stable but unable to be translated in the extract. The circularization was competitively inhibited in the presence of a known substrate of a wheat embryo RNA ligase. Thus, we cloned the RNA ligase cDNAs. Three isoform sequences were homologous to the other plant RNA ligases. An addition of a cell-free synthesized wheat RNA ligase abolished the inhibition, which indicates a participation of its activity in the circularization. A possible role in RNA metabolism, RNA silencing in particular, is discussed.     

Direct Absorption of Methyl Mercury by Lymph

Methyl mercury is contained in fish and seafood products and is taken up into the body in food. While the central nervous system is known as a target organ, methyl mercury also induces autoimmunity and acts as a potent immunosuppressor. The aim of the present study is to know whether methyl mercury is directly absorbed by lymph. Conscious rats were infused with methyl mercury (4 mg/kg) via duodenal tubing as a single pulse infusion, followed by the continuous infusion of saline, and lymphatic fluids were continuously collected from the thoracic lymph duct every 30 min until 360 min after infusion. Mercury was detected immediately after infusion, and total mercury contents in lymph gradually increased until 90–120 min, remained steady, and then gradually decreased until 360 min; however, the amount of mercury collected during 330–360 min was about twofold higher than during 0–30 min. The amount of cumulative mercury in lymph at 360 min was 1.4 μg. In contrast, blood mercury concentration was 2.4 μg/ml 5 min after infusion, with the value at 360 min being 12.6 times higher than at 5 min. Plasma mercury concentration was 56 ng/ml at 5 min, with hundreds of nanograms per milliliter of mercury detected until 360 min. From the present study, it is concluded that some methyl mercury is directly absorbed by lymph and remains steady 6 h after infusion.     

ppGpp inhibits peptide elongation cycle of chloroplast translation system in vitro

Chloroplasts possess common biosynthetic pathways for generating guanosine 3',5'-(bis)pyrophosphate (ppGpp) from GDP and ATP by RelA-SpoT homolog enzymes. To date, several hypothetical targets of ppGpp in chloroplasts have been suggested, but they remain largely unverified. In this study, we have investigated effects of ppGpp on translation apparatus in chloroplasts by developing in vitro protein synthesis system based on an extract of chloroplasts isolated from pea (Pisum sativum). The chloroplast extracts showed stable protein synthesis activity in vitro, and the activity was sensitive to various types of antibiotics. We have demonstrated that ppGpp inhibits the activity of chloroplast translation in dose-effective manner, as does the toxic nonhydrolyzable GTP analog guanosine 5'-(β,γ-imido)triphosphate (GDPNP). We further examined polyuridylic acid-directed polyphenylalanine synthesis as a measure of peptide elongation activity in the pea chloroplast extract. Both ppGpp and GDPNP as well as antibiotics, fusidic acid and thiostrepton, inhibited the peptide elongation cycle of the translation system, but GDP in the similar range of the tested ppGpp concentration did not affect the activity. Our results thus show that ppGpp directly affect the translation system of chloroplasts, as they do that of bacteria. We suggest that the role of the ppGpp signaling system in translation in bacteria is conserved in the translation system of chloroplasts.     

Exploration of a Possible Partnership among Orphan Two-Component System Proteins in Cyanobacterium Synechococcus elongatus PCC 7942

To understand the induction of the adaptive response under various stress conditions, it is important to determine the partnership between histidine kinase and response regulators in the bacterial two-component system (TCS). The genes encoding TCS partners are usually comprised of an operon in the genome, but many of them are orphans in the cyanobacterial genome. There is little information on their partnerships in Synechococcus elongatus PCC 7942. Our comprehensive analysis of protein-protein interactions among all 37 full-length proteins and the truncated domains of 24 orphans revealed a number of specific interactions. They involved evolutionarily well-conserved orphan proteins among cyanobacterial species such as Synpcc7942_0453/Ycf29, NblS/RpaB, NblS/SrrA, SasA/RpaA, and SasA/Synpcc7942_2466. Our investigation of the transphosphorylation of interaction partners indicates that orphan TCSs comprise a complex signaling network.     

Flavonoid metabolism: the interaction of metabolites and gut microbiota

Abstract

Several dietary flavonoids exhibit anti-oxidative, anti-inflammatory, and anti-osteoporotic activities relevant to prevention of chronic diseases, including lifestyle-related diseases. Dietary flavonoids (glycoside forms) are enzymatically hydrolyzed and absorbed in the intestine, and are conjugated to their glucuronide/sulfate forms by phase II enzymes in epithelial cells and the liver. The intestinal microbiota plays an important role in the metabolism of flavonoids found in foods. Some specific products of bacterial transformation, such as ring-fission products and reduced metabolites, exhibit enhanced properties. Studies on the metabolism of flavonoids by the intestinal microbiota are crucial for understanding the role of these compounds and their impact on our health. This review focused on the metabolic pathways, bioavailability, and physiological role of flavonoids, especially metabolites of quercetin and isoflavone produced by the intestinal microbiota.     

Mulberry leaf powder prevents atherosclerosis in apolipoprotein E-deficient mice

Mulberry is commonly used to feed silkworms. Here we examined whether a dietary intake of mulberry leaf (ML) could affect atherogenesis in vivo and in vitro. Apolipoprotein E-deficient mice were fed either normal chow (control group) or a diet containing 1% ML powder (ML group) from 6 weeks of age. The mice were sacrificed after 12 weeks. The susceptibility of plasma lipoprotein to oxidation was assessed using diene formation. A significant increase in the lag time of lipoprotein oxidation was detected in the ML group compared with the control group. Furthermore, the ML group showed a 40% reduction in atherosclerotic lesion size in the aortae compared with the control. We also examined the direct anti-oxidative activity of ML in vitro. Aqueous extract of ML had a strong scavenging effect on 1,1-diphenyl-2-picrylhydrazyl and inhibited lipoprotein oxidation. These results confirm that ML contains anti-oxidative substances that might help prevent atherosclerosis.     

Expression of parsley flavone synthase I establishes the flavone biosynthetic pathway in Arabidopsis thaliana

Arabidopsis thaliana lacks the flavone biosynthetic pathway, probably because of a lack or low activity of a flavone synthase. To establish this biosynthetic pathway in Arabidopsis, we subjected this model plant to transformation with the parsley gene for flavone synthase type I (FNS-I). Transgenic seedlings expressing FNS-I were cultured in liquid medium with or without naringenin, and plant extracts were then analyzed by high-performance liquid chromatography. In contrast to wild-type seedlings, the transgenic seedlings accumulated substantial amounts of apigenin, which is produced from naringenin by FNS-I, and the apigenin level correlated with the abundance of FNS-I mRNA in three different transgenic lines. These results indicate that the FNS-I transgene produces a functional enzyme that catalyzes the conversion of naringenin to apigenin in Arabidopsis. These FNS-I transgenic lines should prove useful in investigating the in vivo functions of enzymes that mediate the synthesis of the wide variety of flavones found in other plants.