A circadian and an ultradian rhythm are both evident in root growth of rice

This paper presents evidence for the existence of both a circadian and an ultradian rhythm in the elongation growth of rice roots. Root elongation of rice (Oryza sativa) was recorded under dim green light by using a CCD camera connected to a computer. Four treatment conditions were set-up to investigate the existence of endogenous rhythms: 28 °C constant temperature and continuous dark (28 DD); 28 °C constant temperature and alternating light and dark (28 LD); 33 °C constant temperature and continuous dark (33 DD); and diurnal temperature change and alternating light and dark (DT-LD). The resulting spectral densities suggested the existence of periodicities of 20.4-25.2 h (circadian cycles) and 2.0-6.0 h (ultradian cycles) in each of the 4 treatments. The shorter ultradian cycles can be attributed to circumnutational growth of roots and/or to mucilage exudation. The average values across all the replicate data showed that the highest power spectral densities (PSDs) corresponded to root growth rhythms with periods of 22.9, 23.7, and 2.1 h for the 28 DD, 28 LD, and 33 DD treatments, respectively. Accumulation of PSD for each data set indicated that the periodicity was similar in both the 28 DD and 33 DD treatments. We conclude that a 23-h circadian and a 2-h ultradian rhythmicity exist in rice root elongation. Moreover, root elongation rates during the day were 1.08 and 1.44 times faster than those during the night for the 28 LD and DT-LD treatments, respectively.     

Successful expression of a novel bacterial gene for pinoresinol reductase and its effect on lignan biosynthesis in transgenic Arabidopsis thaliana

Pinoresinol reductase and pinoresinol/lariciresinol reductase play important roles in an early step of lignan biosynthesis in plants. The activities of both enzymes have also been detected in bacteria. In this study, pinZ, which was first isolated as a gene for bacterial pinoresinol reductase, was constitutively expressed in Arabidopsis thaliana under the control of the cauliflower mosaic virus 35S promoter. Higher reductive activity toward pinoresinol was detected in the resultant transgenic plants but not in wild-type plant. Principal component analysis of data from untargeted metabolome analyses of stem, root, and leaf extracts of the wild-type and two independent transgenic lines indicate that pinZ expression caused dynamic metabolic changes in stems, but not in roots and leaves. The metabolome data also suggest that expression of pinZ influenced the metabolisms of lignan and glucosinolates but not so much of neolignans such as guaiacylglycerol-8-O-4′-feruloyl ethers. In-depth quantitative analysis by liquid chromatography–tandem mass spectrometry (LC-MS/MS) indicated that amounts of pinoresinol and its glucoside form were markedly reduced in the transgenic plant, whereas the amounts of glucoside form of secoisolariciresinol in transgenic roots, leaves, and stems increased. The detected levels of lariciresinol in the transgenic plant following β-glucosidase treatment also tended to be higher than those in the wild-type plant. Our findings indicate that overexpression of pinZ induces change in lignan compositions and has a major effect not only on lignan biosynthesis but also on biosynthesis of other primary and secondary metabolites.     

Characterization of the catabolic pathway for a phenylcoumaran-type lignin-derived biaryl in Sphingobium sp. strain SYK-6

Sphingobium sp. strain SYK-6 is capable of degrading various lignin-derived biaryls. We determined the catabolic pathway of a phenylcoumaran-type compound, dehydrodiconiferyl alcohol (DCA) in SYK-6, and identified some of the DCA catabolism genes. In SYK-6 cells, the alcohol group of DCA was oxidized to the carboxyl group, first at the B-ring side chain and then at the A-ring side chain. The resultant metabolite was degraded to 5-formylferulate and vanillin through the decarboxylation and the Cα–Cβ cleavage of the A-ring side chain. Based on the DCA catabolic pathway, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) genes are thought to be involved in the conversion of DCA into an aldehyde intermediate (DCA-L) and the conversion of DCA-L into a carboxylic acid intermediate (DCA-C), respectively. SLG_05620 and SLG_24930, which belong to quinohemoprotein ADH and aryl ADH, respectively, were isolated as the genes responsible for the oxidation of DCA. In addition to these genes, multiple genes similar to SLG_05620 and SLG_24930 were found to confer DCA oxidation activities on Escherichia coli cells. In order to identify the DCA-L dehydrogenase genes, the DCA-L oxidation activities of the SYK-6 gene products of putative twenty-one ALDH genes were examined. Significant activities were observed in the four ALDH gene products, including the SLG_27910 product, which showed the highest activity. The disruption of SLG_27910 caused a decreased conversion of DCA-L, suggesting that SLG_27910 plays an important role in the DCA-L oxidation. In conclusion, no specific gene seems to be solely responsible for the conversion of DCA and DCA-L, however, the multiple genes encoding quinohemoprotein ADH and aryl ADH genes, and four ALDH genes are probably involved in the conversion processes.     

Lipopolysaccharide Decreases Single Immunoglobulin Interleukin-1 Receptor-related Molecule (SIGIRR) Expression by Suppressing Specificity Protein 1 (Sp1) via the Toll-like Receptor 4 (TLR4)-p38 Pathway in Monocytes and Neutrophils

Single immunoglobulin interleukin-1 receptor-related molecule (SIGIRR) is one of the immunoglobulin-like membrane proteins that is crucial for negative regulation of toll-like receptor 4 (TLR4) and interleukin-1 receptor. Despite the importance of understanding its expression and function, knowledge is limited on the regulatory mechanism in the epithelial tissues, such as the liver, lung, and gut, where its predominant expression is originally described. Here, we found expression of SIGIRR in non-epithelial innate immune cells, including primary peripheral blood monocytes, polymorphonuclear neutrophils, monocytic RAW264 cells, and neutrophilic-differentiated HL-60 cells. Consistent with previous findings in epithelial tissues, SIGIRR gene and protein expression were also down-regulated by LPS treatment in a time-dependent manner in primary blood monocytes and polymorphonuclear neutrophils. A reduction was also observed in RAW264 and differentiated HL-60 cells. Notably, exogenous introduction of the dominant negative form of TLR4 and siRNA of p38 resulted in inhibition of LPS-induced SIGIRR down-regulation, whereas treatment with p38 activator anisomycin showed a dose-dependent decrease in SIGIRR expression, suggesting TLR4-p38 signal as a critical pathway for LPS-induced SIGIRR down-regulation. Finally, reporter gene and chromatin immunoprecipitation assays demonstrated that Sp1 is a key factor that directly binds to the proximal promoter of SIGIRR gene and consequently regulates basal SIGIRR expression, which is negatively regulated by the LPS-dependent TLR4-p38 pathway. In summary, the data precisely demonstrate how LPS down-regulates SIGIRR expression and provide a role of LPS signal that counteracts Sp1-dependent basal promoter activation of SIGIRR gene via TLR4-p38 pathway in non-epithelial innate immune cells.     

Fucosylation of N-glycans regulates the secretion of hepatic glycoproteins into bile ducts

Fucosylated alpha-fetoprotein (AFP) is a highly specific tumor marker for hepatocellular carcinoma (HCC). However, the molecular mechanism by which serum level of fucosylated AFP increases in patients with HCC remains largely unknown. Here, we report that the fucosylation of glycoproteins could be a possible signal for secretion into bile ducts in the liver. We compared oligosaccharide structures on glycoproteins in human bile with those in serum by several types of lectin blot analyses. Enhanced binding of biliary glycoproteins to lectins that recognize a fucose residue was observed over a wide range of molecular weights compared with serum glycoproteins. A structural analysis of oligosaccharides by two-dimensional mapping high performance liquid chromatography and matrix-assisted laser desorption ionization time-of flight mass spectrometry confirmed the increases in the fucosylation of biliary glycoproteins. Purification followed by structural analysis on alpha1-antitrypsin, alpha1-acid glycoprotein and haptoglobin, which are synthesized in the liver, showed higher fucosylation in bile than in serum. To find direct evidence for fucosylation and sorting signal into bile ducts, we used alpha1-6 fucosyltransferase (Fut8)-deficient mice because fucosylation of glycoproteins produced in mouse liver was mainly an alpha1-6 linkage. Interestingly, the levels of alpha1-antitrypsin and alpha1-acid glycoprotein were quite low in bile of Fut8-deficient mice as compared with wild-type mice. An immunohistochemical study showed dramatic changes in the localization of these glycoproteins in the liver of Fut8-deficient mice. Taken together, these results suggest that fucosylation is a possible signal for the secretion of glycoproteins into bile ducts in the liver. A disruption in this system might involve an increase in fucosylated AFP in the serum of patients with HCC.     

Development and characterisation of microsatellite loci in Farfugium japonicum (Asteraceae)

Eight microsatellite loci for the perennial herb Farfugium japonicum, including the rheophytic variety luchuense endemic to riparian areas of the Ryukyu Islands, Japan, were isolated and characterised. The number of alleles ranged from 5 to 14. The expected (H _E) and observed (H _O) heterozygosities were 0.344–0.885 and 0.121–0.754, respectively, from 69 individuals in one population. Six loci exhibited significantly fewer heterozygotes than expected under Hardy–Weinberg equilibrium (P < 0.05). The primers amplifying microsatellite sequences in F. japonicum may provide a population genetics tool useful in the establishment of a conservation strategy.     

High yield secretion of the sweet-tasting protein lysozyme from the yeast Pichia pastoris

Hen egg lysozyme (HEL) is one of the sweet-tasting proteins. To understand why lysozyme is sweet, the enzyme was synthesized at high yields by a recombinant method. The mature HEL gene was cloned from a Taq polymerase-amplified PCR product into the Pichia pastoris expression and secretion vector pPIC6alpha. This expression vector contains both the Saccharomyces cerevisiae pre-pro alpha-mating factor secretion signal and the blasticidin resistance gene (bsd) for selection of transformants in bacteria and yeast. Expression of HEL was carried out in fermenter cultures. Culture supernatants were concentrated by ultrafiltration and purified by CM-ion exchange chromatography. Approximately 400 mgL-1 of recombinant HEL was obtained. The high yield of recombinant lysozyme enabled us to perform a sensory analysis in humans. The purified recombinant lysozyme elicited as a sweet taste sensation as does the lysozyme purified directly from egg white, and showed full lytic activity against cells of Micrococcus luteus. These results demonstrate that the P. pastoris expression system with the blasticidin S selection system is useful in producing recombinant sweet-tasting protein in active form at a high yield.     

Two novel decarboxylase genes play a key role in the stereospecific catabolism of dehydrodiconiferyl alcohol in Sphingobium sp. strain SYK‐6

Sphingobium sp. strain SYK‐6 is able to use a phenylcoumaran‐type biaryl, dehydrodiconiferyl alcohol (DCA), as a sole source of carbon and energy. In SYK‐6 cells, the alcohol group of the B‐ring side chain of DCA was first oxidized to the carboxyl group, and then the alcohol group of the A‐ring side chain was oxidized to generate 5‐(2‐carboxyvinyl)‐2‐(4‐hydroxy‐3‐methoxyphenyl)‐7‐methoxy‐2,3‐dihydrobenzofuran‐3‐carboxylate (DCA‐CC). We identified phcF, phcG and phcH, which conferred the ability to convert DCA‐CC into 3‐(4‐hydroxy‐3‐(4‐hydroxy‐3‐methoxystyryl)‐5‐methoxyphenyl)acrylate (DCA‐S) in a host strain. These genes exhibited no significant sequence similarity with known enzyme genes, whereas phcF and phcG, which contain a DUF3237 domain of unknown function, showed 32% amino acid sequence identity with each other. The DCA‐CC conversion activities were markedly decreased by disruption of phcF and phcG, indicating that phcF and phcG play dominant roles in the conversion of DCA‐CC. Purified PhcF and PhcG catalysed the decarboxylation of the A‐ring side chain of DCA‐CC, producing DCA‐S, and showed enantiospecificity towards (+)‐ and (–)‐DCA‐CC respectively. PhcF and PhcG formed homotrimers, and their K_m for DCA‐CC were determined to be 84 μM and 103 μM, and V_max were 307 μmol?min^?1?mg^?1 and 137 μmol?min^?1?mg^?1 respectively. In conclusion, PhcF and PhcG are enantiospecific decarboxylases involved in phenylcoumaran catabolism.     

Signalling pathway of tumor necrosis factor in normal and tumor cells

Summary Several aspects of the activity and effects of tumor necrosis factor (TNF) were investigated to gain further insight into its cytotoxic mechanism. The relation between number of TNF receptors and TNF susceptibility of both tumor cells and normal cells was studied, utilizing a specific binding assay. Among the tumor cells, a fairly close correlation (r=0.855) was observed between receptor number and sensitivity to TNF. No cytotoxic effect by TNF was observed on any of the normal cells tested, even though TNF receptors were shown to be present, and cell proliferation was apparently stimulated by TNF in some cases. TNF internalization and intracellular distribution were studied by pulse-labelling and Percoll density gradient centrifugation. In L-M (murine tumorigenic fibroblasts, highly sensitive to TNF cytotoxicity) cells and HEL (human embryonic lung cells, non-sensitive to TNF cytotoxicity) cells, receptor-bound ¹²⁵I-labelled recombinant human TNF was rapidly internalized and delivered to lysosomes within 15–30 min, and this was followed by degradation and release into the culture medium. The presence of either a cytoskeletal disrupting agent or a lysosomotropic agent was observed to inhibit the cytotoxic effect of TNF, thus also indicating that TNF internalization, followed by delivery to lysosomes, is essential in the cytolytic mechanism of TNF.As observed by [³H]uridine incorporation, TNF did not affect RNA synthesis in L-R cells (TNF-resistant cell lines derived from L-M cells) and HEL cells, but markedly stimulated (by 3.5 times) RNA synthesis in L-M cells.