cDNA cloning and biochemical characterization of S-adenosyl-L-methionine: 2,7,4'-trihydroxyisoflavanone 4'-O-methyltransferase,a critical enzyme of the legume isoflavonoid phytoalexin pathway

Formononetin (7-hydroxy-4'-methoxyisoflavone, also known as 4'-O-methyldaidzein) is an essential intermediate of ecophysiologically active leguminous isoflavonoids. The biosynthetic pathway to produce 4'-methoxyl of formononetin has been unknown because the methyl transfer from S-adenosyl-L-methionine (SAM) to 4'-hydroxyl of daidzein has never been detected in any plants. A hypothesis that SAM: daidzein 7-O-methyltransferase (D7OMT), an enzyme with a different regiospecificity, is involved in formononetin biosynthesis through its intracellular compartmentation with other enzymes recently prevails, but no direct evidence has been presented. We proposed a new scheme of formononetin biosynthesis involving 2,7,4'-trihydroxyisoflavanone as the methyl acceptor and subsequent dehydration. We now cloned a cDNA encoding SAM: 2,7,4'-trihydroxyisoflavanone 4'-O-methyltransferase (HI4'OMT) through the screening of functionally expressed Glycyrrhiza echinata (Fabaceae) cDNAs. The reaction product, 2,7-dihydroxy-4'-methoxyisoflavanone, was unambiguously identified. Recombinant G. echinata D7OMT did not show HI4'OMT activity, and G. echinata HI4'OMT protein free from D7OMT was partially purified. HI4'OMT is thus concluded to be distinct from D7OMT, and their distant phylogenetic relationship was further presented. HI4'OMT may be functionally identical to (+)-6a-hydroxymaackiain 3-OMT of pea. Homologous cDNAs were found in several legumes, and the catalytic function of the Lotus japonicus HI4'OMT was verified, indicating that HI4'OMT is the enzyme of formononetin biosynthesis in general legumes.     

Direct analysis of drugs in plasma by column-switching liquid chromatography-mass spectrometry using a methylcellulose-immobilized reversed-phase pretreatment column

A novel methylcellulose-immobilized reversed-phase pretreatment column (MC-ODS) for column switching liquid chromatography-mass spectrometry (LC-MS) was investigated to improve recovery and durability. Pretreatment and analytical conditions were optimized so that high throughput and high selectivity was ensured during mass spectrometric analysis. Analytical runs, including deproteinization and gradient LC analysis, were conducted in a 6-min cycle. As a consequence, recoveries for test drugs (metoprolol, propranolol, lidocaine, dibucaine, bupivacaine) were greater than 90% and more than 300 plasma samples spiked with target compounds were directly injected and measured without compromising MS detection or system performance.     

Crystal structures of bacterial lipoprotein localization factors,LolA and LolB

Lipoproteins having a lipid-modified cysteine at the N-terminus are localized on either the inner or the outer membrane of Escherichia coli depending on the residue at position 2. Five Lol proteins involved in the sorting and membrane localization of lipoprotein are highly conserved in Gram-negative bacteria. We determined the crystal structures of a periplasmic chaperone, LolA, and an outer membrane lipoprotein receptor, LolB. Despite their dissimilar amino acid sequences, the structures of LolA and LolB are strikingly similar to each other. Both have a hydrophobic cavity consisting of an unclosed beta barrel and an alpha-helical lid. The cavity represents a possible binding site for the lipid moiety of lipoproteins. Detailed structural differences between the two proteins provide significant insights into the molecular mechanisms underlying the energy-independent transfer of lipoproteins from LolA to LolB and from LolB to the outer membrane. Furthermore, the structures of both LolA and LolB determined from different crystal forms revealed the distinct structural dynamics regarding the association and dissociation of lipoproteins. The results are discussed in the context of the current model for the lipoprotein transfer from the inner to the outer membrane through a hydrophilic environment.     

Neurogenesis and the cell cycle

For a long time, it has been understood that neurogenesis is linked to proliferation and thus to the cell cycle. Recently, the gears that mediate this linkage have become accessible to molecular investigation. This review describes some of the progress that has been made in understanding how the molecular machinery of the cell cycle is used in the processes of size regulation in the brain, histogenesis, neuronal differentiation, and the maintenance of stem cells.     

Changes in cell volume of bacteria and heterotrophic nanoflagellates in a hypereutrophic pond

Changes in cell volume of planktonic bacteria and heterotrophic nanoflagellates (HNF) were examined in a hypereutrophic pond from April to October, 1997. There were marked changes in the abundance of bacteria, HNF and ciliates and in protistan bacterivory during this period. The cell volume of free-living bacteria (0.121 ± 0.031 m³, mean ± SD) was large relative to that reported in the literature. The cell volumes of HNF was 71.1 ± 24.8 m³. Both cell volumes did not follow a seasonal trend. The dominant size class of bacteria was seasonally variable, whereas density of filamentous bacteria was relatively high between August and September. Biomass of filamentous bacteria accounted for up to 33.6% of total bacterial biomass. A correlation analysis for cell volume of bacteria and HNF, density of filamentous bacteria and some microbial variates was performed. The positive correlations detected (p<0.05) were between density of bacteria and cell volume of HNF, and between density of filamentous bacteria and cell volume of HNF.     

A comparison of solid and liquid media for resuscitation of starvation- and low-temperature-induced nonculturable cells of Aeromonas hydrophila

Like many other gram-negative bacteria, starved cells of Aeromonas hydrophila can be induced into a viable but nonculturable (VBNC) state by incubation at low temperature, as shown here by using various bacterial enumeration methods. Starved A. hydrophila strain HR7 cells at 4 degrees C reached the nonculturable stage in about 45 days. The cells were resuscitated by either a solid medium resuscitation method, using solid agar amended with H2O2-degrading agents, catalase or sodium pyruvate, or a liquid medium resuscitation method, by incubating nonculturable cells in liquid media containing these compounds before spreading onto plates. The liquid medium resuscitation method using catalase resulted in nearly complete recovery of nonculturable cells.     

Rhododendrol biosynthesis and metabolism in Acer nikoense callus

Cell suspensions derived from Acer nikoense callus, not containing (S)-rhododendrol, converted 4-(p-hydroxyphenyl)-2- butanone into (R)-, (S)-rhododendrol and their glycosides. (R)- and (S)-rhododendrol formed was only detected in the culture medium and their glycosides only in the cells. The former compound disappeared within a short time and the latter one also tended to decrease during prolonged culture. Quantitative analysis of rhododendrol glycosides in the callus showed that most of them were (S)-rhododendrol 2-O--D-glucopyranoside and its content was much lower than that of the original plants. © Rapid Science Ltd. 1998     

Changes in brain metallothionein and Zinc during development in transgenic mice

The developmental alterations in metallothionein (MT) proteins and zinc (Zn) were investigated in brains of two transgenic strains of mice. MT protein was measured by a cadmium binding assay and Zn by atomic absorption spectrophotometry. MT proteins were expressed at birth (day 1) both in MT-I overexpressing transgenic mouse (MT-I*) and MT-null (expressing only brain specific isoform, MT-III) transgenic mouse. MT proteins level (mainly MT-I) in MT-I* was 16.1 Μ-g/g at birth, and thereafter increased with age to a maximal adult level of 55.3 Μg/g (day 60). Zn level in MT-I* also increased from 8.43 Μg/g (day 1) to 20.7 Μg/g (day 60) with age. MT protein (MT-III) in MT-null mouse was 9.71 Μg/g at birth and remained relatively unchanged during development. Zn level in MT-null mouse at birth was 9.46 Μg/g and also remained unchanged during development. The similar alterations in MT isoforms and Zn in brain during development suggest that MT isoforms may act as a Zn binding protein.     

Induction of Neurite Outgrowth in PC12 Cells Treated with Temperature-Controlled Repeated Thermal Stimulation

To promote the functional restoration of the nervous system following injury, it is necessary to provide optimal extracellular signals that can induce neuronal regenerative activities, particularly neurite formation. This study aimed to examine the regulation of neuritogenesis by temperature-controlled repeated thermal stimulation (TRTS) in rat PC12 pheochromocytoma cells, which can be induced by neurotrophic factors to differentiate into neuron-like cells with elongated neurites. A heating plate was used to apply thermal stimulation, and the correlation of culture medium temperature with varying surface temperature of the heating plate was monitored. Plated PC12 cells were exposed to TRTS at two different temperatures via heating plate (preset surface temperature of the heating plate, 39.5°C or 42°C) in growth or differentiating medium for up to 18 h per day. We then measured the extent of growth, neuritogenesis, or acetylcholine esterase (AChE) activity (a neuronal marker). To analyze the mechanisms underlying the effects of TRTS on these cells, we examined changes in intracellular signaling using the following: tropomyosin-related kinase A inhibitor GW441756; p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580; and MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor U0126 with its inactive analog, U0124, as a control. While a TRTS of 39.5°C did not decrease the growth rate of cells in the cell growth assay, it did increase the number of neurite-bearing PC12 cells and AChE activity without the addition of other neuritogenesis inducers. Furthermore, U0126, and SB203580, but not U0124 and GW441756, considerably inhibited TRTS-induced neuritogenesis. These results suggest that TRTS can induce neuritogenesis and that participation of both the ERK1/2 and p38 MAPK signaling pathways is required for TRTS-dependent neuritogenesis in PC12 cells. Thus, TRTS may be an effective technique for regenerative neuromedicine.     

IL-38: A new factor in rheumatoid arthritis

The newly characterized cytokine IL-38 (IL-1F10) belongs to the IL-1 family of cytokines. Previous work has demonstrated that IL-38 inhibited Candida albicans-induced IL-17 production from peripheral blood mononuclear cells. However, it is still unclear whether IL-38 is an inflammatory or an anti-inflammatory cytokine. We generated anti-human IL-38 monoclonal antibodies in order to perform immunohistochemical staining and an enzyme-linked immunosorbent assay. While human recombinant IL-38 protein was not cleaved by recombinant caspase-1, chymase, or PR3 in vitro, overexpression of IL-38 cDNA produced a soluble form of IL-38 protein. Furthermore, immunohistochemical analysis showed that synovial tissues obtained from RA patients strongly expressed IL-38 protein. To investigate the biological role of IL-38, C57BL/6 IL-38 gene-deficient (^?/?) mice were used in an autoantibody-induced rheumatoid arthritis (RA) mouse model. As compared with control mice, IL-38 ^(?/?) mice showed greater disease severity, accompanied by higher IL-1β and IL-6 gene expression in the joints. Therefore, IL-38 acts as an inhibitor of the pathogenesis of autoantibody-induced arthritis in mice and may have a role in the development or progression of RA in humans.