Expression analysis of cellulose synthases that comprise the Type II complex in hybrid aspen.

Gene duplication in plants occurs via several different mechanisms, including whole genome duplication, and the copied genes acquire various forms and types. The cellulose synthase (CesA) family functions in cellulose synthesis complex (CSC) formation, which is involved in the synthesis of primary and secondary cell walls in plants. In the genome of Populus, 17 CesA have been annotated, and some of them appeared through whole genome duplication. The nucleotide sequence of the duplicated genes changed during subsequent evolution, and functional differentiation of genes might have occurred. To gain insight into the evolutionary fate of the duplicated CesA, expression analysis with quantitative reverse transcription polymerase chain reactions and promoter-reporter assays was performed on three duplicated gene pairs whose products have been reported to form a single CSC. Changes in expression of each gene at different developmental stages were detected and divergent expression patterns in different organs and tissues observed between the gene pairs. Among the tested genes, expression of PttCesA3-C was apparently lower than that of its counterpart, PttCesA3-D. The results suggest that the six CesA are approaching sub-functionalisation or non-functionalisation. Furthermore, the level of functionalisation may vary among the three pairs of genes, and functional specialisation of each CesA should have been achieved, at least partially, through differences in expression of genes.

Development of apertureless near-field scanning optical microscope tips for tip-enhanced Raman spectroscopy.

In this study, we suggested two types of novel metallized tip for the apertureless near-field scanning optical microscope probe. The first is a silver nanorod immobilized tip and the other is a double metallized probe. We calculated the electric field enhancement factor and the electric field distribution of a single sphere, aggregated spheres, an ellipse and a nanorod by the finite-differential time-domain method to improve the silver nanosphere immobilized tip developed in our previous studies. The enhanced field of the nanorod is localized at the external surfaces. The simulation results of the nanorod revealed that the position of the maximum peak is shifted to a longer wavelength and that its electric field enhancement factor increases as the aspect ratio increases. Thus, we developed the silver nanorod immobilized tip, and the tip-enhanced Raman spectrum of rhodamine 6G molecule on the substrate could be measured by the tip though it could not be detected by the previous nanosphere immobilized tip. Further, the finite-differential time-domain calculation predicted that the double metallized tips considerably enhance the electric field and that its enhancement factor in the longer wavelength region (500-600 nm) does not decrease when the tip is rounded. The results show that the proposed metallized tips were useful for the apertureless near-field scanning optical microscope system.

Progress of lignification mediated by intercellular transportation of monolignols during tracheary element differentiation of isolated Zinnia mesophyll cells.

Tracheary element (TE) differentiation is a typical example of programmed cell death (PCD) in higher plants, and maturation of TEs is completed by degradation of all cell contents. However, lignification of TEs progresses even after PCD. We investigated how and whence monolignols are supplied to TEs which have undergone PCD during differentiation of isolated Zinnia mesophyll cells into TEs. Higher densities of cell culture induced greater lignification of TEs. Whereas the continuous exchanging of culture medium suppressed lignification of TEs, further addition of coniferyl alcohol into the exchanging medium reduced the suppression of lignification. Analysis of the culture medium by HPLC and GC-MS showed that coniferyl alcohol, coniferaldehyde, and sinapyl alcohol accumulated in TE inductive culture. The concentration of coniferyl alcohol peaked at the beginning of secondary wall thickening, decreased rapidly during secondary wall thickening, then increased again. These results indicated that lignification on TEs progresses by supply of monolignols from not only TEs themselves but also surrounding xylem parenchyma-like cells through medium in vitro.

A case of undifferentiated glioma in a 70-year-old woman.

The present case involved a 70-year-old woman who was diagnosed with a right cerebral hemorrhage. Excisional surgery of the hematoma was performed. Grossly, a whitish, solid tumor (1 x 1 x 0.8 cm in size) was recognized in the hematoma. Histologically, the tumor was composed of large, polygonal cells and small undifferentiated cells in a jumbled architectural arrangement with a cartilage component. The large, polygonal cell component was conspicuous and somewhat rhabdoid in appearance and appeared to be an astrocytic tumor showing glial differentiation. The small, undifferentiated cell component resembled tumor cells of a primitive neuroectodermal tumor (PNET). Clinical follow-up of the patient for 2 months after the first operation revealed recurrence with rapid growth. A second operation was performed, but the patient died 8 months after the first operation (2 months after the second). Immunohistochemically, the tumor cells suggesting glial differentiation were positive for glial fibrillary acidic protein (GFAP), S-100, neuron-specific enolase (NSE), and vimentin. PNET-like components in the primary tumor were positive for NSE, GFAP, and S-100, and weakly positive for vimentin and synaptophysin. Each tumor cell was negative for epithelial membrane antigen (EMA), keratin, desmin, actin, myoglobin, neurofilament (NF), and MIC2 protein. The recurrent tumor revealed predominantly PNET-like components; however, only a few tumor cells were positive for GFAP. This appearance suggested that this brain tumor might originate from a common multipotential stem cell. Considering its histopathological and immunohistochemical characteristics, the primary tumor was finally regarded as an undifferentiated glioma with dedifferentiation of the glial component in the recurrent tumor.

The last step of syringyl monolignol biosynthesis in angiosperms is regulated by a novel gene encoding sinapyl alcohol dehydrogenase.

Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) has been thought to mediate the reduction of both coniferaldehyde and sinapaldehyde into guaiacyl and syringyl monolignols in angiosperms. Here, we report the isolation of a novel aspen gene (PtSAD) encoding sinapyl alcohol dehydrogenase (SAD), which is phylogenetically distinct from aspen CAD (PtCAD). Liquid chromatography-mass spectrometry-based enzyme functional analysis and substrate level-controlled enzyme kinetics consistently demonstrated that PtSAD is sinapaldehyde specific and that PtCAD is coniferaldehyde specific. The enzymatic efficiency of PtSAD for sinapaldehyde was approximately 60 times greater than that of PtCAD. These data suggest that in addition to CAD, discrete SAD function is essential to the biosynthesis of syringyl monolignol in angiosperms. In aspen stem primary tissues, PtCAD was immunolocalized exclusively to xylem elements in which only guaiacyl lignin was deposited, whereas PtSAD was abundant in syringyl lignin-enriched phloem fiber cells. In the developing secondary stem xylem, PtCAD was most conspicuous in guaiacyl lignin-enriched vessels, but PtSAD was nearly absent from these elements and was conspicuous in fiber cells. In the context of additional protein immunolocalization and lignin histochemistry, these results suggest that the distinct CAD and SAD functions are linked spatiotemporally to the differential biosynthesis of guaiacyl and syringyl lignins in different cell types. SAD is required for the biosynthesis of syringyl lignin in angiosperms.

Influence of natural and synthetic compounds on cell surface expression of cell adhesion molecules, ICAM-1 and VCAM-1.

Various natural and synthetic compounds including alkaloids, terpenoids and phenolics were tested for inhibition of the cell surface expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), both of which are crucial in the regulation of immune response and inflammation. Of 40 compounds tested, two compounds significantly downregulated the expression of VCAM-1 on murine endothelial cells (F-2) and ten compounds that of ICAM-1 on mouse myeloid leukemia cells (M1). Sanguinarine chloride (5) and isoliquiritigenin (13) were capable of lowering the levels of both ICAM-1 and VCAM-1. The structure-activity relationships study on chalcone and flavone derivatives related to 13 suggested that the inhibitory activity of the chalcone derivatives is attributable to the 4-hydroxy group as well as the possible coplanarity between the phenyl ring and the adjacent conjugated ketone.

5-hydroxyconiferyl aldehyde modulates enzymatic methylation for syringyl monolignol formation, a new view of monolignol biosynthesis in angiosperms.

S-Adenosyl-L-methionine-dependent caffeate O-methyltransferase (COMT, EC 2.1.1.6) has traditionally been thought to catalyze the methylation of caffeate and 5- hydroxyferulate for the biosynthesis of syringyl monolignol, a lignin constituent of angiosperm wood that enables efficient lignin degradation for cellulose production. However, recent recognition that coniferyl aldehyde prevents 5-hydroxyferulate biosynthesis in lignifying tissue, and that the hydroxylated form of coniferyl aldehyde, 5-hydroxyconiferyl aldehyde, is an alternative COMT substrate, demands a re-evaluation of the role of COMT during monolignol biosynthesis. Based on recombinant aspen (Populus tremuloides) COMT enzyme kinetics coupled with mass spectrometry analysis, this study establishes for the first time that COMT is in fact a 5-hydroxyconiferyl aldehyde O-methyltransferase (AldOMT), and that 5-hydroxyconiferyl aldehyde is both the preferred AldOMT substrate and an inhibitor of caffeate and 5-hydroxyferulate methylation, as measured by K(m) and K(i) values. 5-Hydroxyconiferyl aldehyde also inhibited the caffeate and 5-hydroxyferulate methylation activities of xylem proteins from various angiosperm tree species. The evidence that syringyl monolignol biosynthesis is independent of caffeate and 5-hydroxyferulate methylation supports our previous discovery that coniferyl aldehyde prevents ferulate 5-hydroxylation and at the same time ensures a coniferyl aldehyde 5-hydroxylase (CAld5H)-mediated biosynthesis of 5-hydroxyconiferyl aldehyde. Together, our results provide conclusive evidence for the presence of a CAld5H/AldOMT-catalyzed coniferyl aldehyde 5-hydroxylation/methylation pathway that directs syringyl monolignol biosynthesis in angiosperms.

Comparisons of the pharmacokinetics and the leukopenia and thrombocytopenia grade after administration of irinotecan and 5-fluorouracil in combination to rats.

BACKGROUND: Irinotecan (CPT-11) has been used recently for the treatment of several cancers in combination with 5-fluorouracil (5-FU). Preliminary data on this combination therapy in humans demonstrated no drug interactions between CPT-11 (or its metabolite, SN-38) and 5-FU however, because there is so little information on the combination, the possibility for an interaction still exists. MATERIAL AND METHODS: CPT-11 and 5-FU were injected intravenously into rats and the pharmacokinetics of CPT-11 and SN-38 and alternations in blood cell count were investigated. RESULTS: In the group of rats administered 5-FU prior to CPT-11, the area under the concentration-time curve (AUC) of CPT-11 was approximately eight-fold larger compared with the group administered CPT-11 prior to 5-FU. On the other hand, the grade of leukocytopenia or thrombocytopenia was not significantly different among the two groups. CONCLUSION: In rats, the conversion of CPT-11 into SN-38 is possibly delayed by prior administration of 5-FU.

Coniferyl aldehyde 5-hydroxylation and methylation direct syringyl lignin biosynthesis in angiosperms.

A central question in lignin biosynthesis is how guaiacyl intermediates are hydroxylated and methylated to the syringyl monolignol in angiosperms. To address this question, we cloned cDNAs encoding a cytochrome P450 monooxygenase (LsM88) and a caffeate O-methyltransferase (COMT) from sweetgum (Liquidambar styraciflua) xylem. Mass spectrometry-based functional analysis of LsM88 in yeast identified it as coniferyl aldehyde 5-hydroxylase (CAld5H). COMT expressed in Escherichia coli methylated 5-hydroxyconiferyl aldehyde to sinapyl aldehyde. Together, CAld5H and COMT converted coniferyl aldehyde to sinapyl aldehyde, suggesting a CAld5H/COMT-mediated pathway from guaiacyl to syringyl monolignol biosynthesis via coniferyl aldehyde that contrasts with the generally accepted route to sinapate via ferulate. Although the CAld5H/COMT enzyme system can mediate the biosynthesis of syringyl monolignol intermediates through either route, k(cat)/K(m) of CAld5H for coniferyl aldehyde was approximately 140 times greater than that for ferulate. More significantly, when coniferyl aldehyde and ferulate were present together, coniferyl aldehyde was a noncompetitive inhibitor (K(i) = 0.59 microM) of ferulate 5-hydroxylation, thereby eliminating the entire reaction sequence from ferulate to sinapate. In contrast, ferulate had no effect on coniferyl aldehyde 5-hydroxylation. 5-Hydroxylation also could not be detected for feruloyl-CoA or coniferyl alcohol. Therefore, in the presence of coniferyl aldehyde, ferulate 5-hydroxylation does not occur, and the syringyl monolignol can be synthesized only from coniferyl aldehyde. Endogenous coniferyl, 5-hydroxyconiferyl, and sinapyl aldehydes were detected, consistent with in vivo operation of the CAld5H/COMT pathway from coniferyl to sinapyl aldehydes via 5-hydroxyconiferyl aldehyde for syringyl monolignol biosynthesis.

Changes in quinone profiles of hot spring microbial mats with a thermal gradient

The respiratory and photosynthetic quinones of microbial mats which occurred in Japanese sulfide-containing neutral-pH hot springs at different temperatures were analyzed by spectrochromatography and mass spectrometry. All of the microbial mats that developed at high temperatures (temperatures above 68 degreesC) were so-called sulfur-turf bacterial mats and produced methionaquinones (MTKs) as the major quinones. A 78 degreesC hot spring sediment had a similar quinone profile. Chloroflexus-mixed mats occurred at temperatures of 61 to 65 degreesC and contained menaquinone 10 (MK-10) as the major component together with significant amounts of either MTKs or plastoquinone 9 (PQ-9). The sunlight-exposed biomats growing at temperatures of 45 to 56 degreesC were all cyanobacterial mats, in which the photosynthetic quinones (PQ-9 and phylloquinone) predominated and MK-10 was the next most abundant component in most cases. Ubiquinones (UQs) were not found or were detected in only small amounts in the biomats growing at temperatures of 50 degreesC and above, whereas the majority of the quinones of a purple photosynthetic mat growing at 34 degreesC were UQs. A numerical analysis of the quinone profiles was performed by using the following three parameters: dissimilarity index (D), microbial divergence index (MDq), and bioenergetic divergence index (BDq). A D matrix tree analysis showed that the hot spring mats consisting of the sulfur-turf bacteria, Chloroflexus spp., cyanobacteria, and purple phototrophic bacteria formed distinct clusters. Analyses of MDq and BDq values indicated that the microbial diversity of hot spring mats decreased as the temperature of the environment increased. The changes in quinone profiles and physiological types of microbial mats in hot springs with thermal gradients are discussed from evolutionary viewpoints.

Imaging of optical disc using reflection-mode scattering-type scanning near-field optical microscopy.

A phase-change optical disc was observed using a reflection-mode scattering-type scanning near-field optical microscope (RS-SNOM). In an a.c.-mode SNOM image, the 1.2 microm x 0.6 microm recording marks were successfully observed although the data were recorded on the groove. In contrast, no recording marks could be resolved in a d.c.-mode SNOM image. These results are in good agreement with those from a numerical simulation using the finite difference time domain method. The resolution was better than 100 nm with a.c.-mode SNOM operation and the results indicate that recording marks in phase-change optical media can be directly observed with the RS-SNOM.

Topographical evaluation of optical disks with polycarbonate substrates in the manufacturing process.

An optical contour mapping with a laser-interference method was carried out to evaluate the topographical deformation of optical memory disks. The dynamics of the deformation in a manufacturing process was studied. Reflective films and a protective resin coating increased the dishing, irrespective of the original shape of a substrate. A bonding process decreased the dishing and contributed to mechanical stability that lasted for a long period in daily use. In the case of a 0.6-mm-thick substrate, as with a DVD disk, the large dishing of the single substrate was suppressed by the bonding to less than 100 microm. In addition, it was shown that distortions arising from the molding process remained in the final product and that the initial process should be carefully controlled.

Stereochemical Difference in Secoisolariciresinol Formation between Cell-free Extracts from Petioles and from Ripening Seeds of Arctium lappa L.

Cell-free extracts from ripening seeds of Arctium lappa L. catalyzed the enantioselective formation of (-)-pinoresinol, (-)-lariciresinol and (-)-secoisolariciresinol from achiral coniferyl alcohol in the presence of NADPH and H2O2. The enantioselectivity of the lignan formation was opposite to that of the (+)-secoisolariciresinol formation catalyzed by cell-free extracts from petioles of the same plant species.

Effects of dietary protein restriction on nitrogen balance and cardiovascular functions in aged rats.

Effects of aging and dietary protein restriction on nitrogen balance and cardiovascular functions were examined. Male Fischer 344 rats 6-7 months old were fed ad libitum until 24 or 25 months old with either a 12% or a 23% protein diet, respectively. The nitrogen balance measured at the age of 24 months old demonstrated a significant difference between the two groups: the group with the 23% protein diet had a negative balance, while the group with the 12% protein diet had a positive balance. Endothelium-dependent relaxation with acetylcholine of the thoracic aortas was impaired by age but to a lesser extent to the protein-restricted animals. In addition, increased ability of platelet aggregation according to aging was also significantly suppressed by protein restriction. These observations demonstrate that protein restriction delays the aging effects of nitrogen loss and reduced cardiovascular function.

Purification and partial sequences of Aralia cordata cinnamyl alcohol dehydrogenase.

Cinnamyl alcohol dehydrogenase (CAD) (EC 1.1.1.195) from a dicot, Aralia cordata, was purified to homogeneity and its properties were characterized. The enzyme shows a preference for cinnamyl alcohols and cinnamyl aldehydes as substrates. The M(r) is estimated at 72,000. The enzyme is composed of two heterogeneous subunits of slightly different sizes, and it differs from the bean enzyme in the size of subunits. Partial amino acid sequencing of the purified enzyme was carried out both from the N-terminus and using selected peptides obtained by cyanogen bromide cleavage.

Treatment of Kawasaki disease using the intravenous aspirin anti-inflammatory effect of salicylate.

Serum salicylate concentrations were measured in 60 patients with acute phase Kawasaki disease (KD), who were treated with intravenous aspirin (IVASP), to evaluate its anti-inflammatory effect in the treatment of KD. Patients with serum salicylate concentrations > or = 150 micrograms/ml showed shorter durations of fever (7.1 +/- 2.0 vs 10.4 +/- 6.6 days; P < 0.05), shorter durations of positive serum C-reactive protein (14.6 +/- 4.5 vs 22.3 +/- 10.6 days; P < 0.01) and lower incidences of coronary arterial involvements (0/10 vs 6/24; P < 0.05) than did patients with serum salicylate concentrations < 150 micrograms/ml. Significant linear correlations were recognized between daily IVASP dosage and serum salicylate concentrations (r = 0.73; P < 0.01), and between serum salicylate concentrations and serum free salicylate concentrations (r = 0.82; P < 0.01). These correlations did not differ between the presence and absence of coronary arterial involvements. Based on these findings we concluded that a beneficial anti-inflammatory effect in the treatment of KD is achieved when the serum salicylate concentration is > or = 150 micrograms/ml, and that such concentrations could be achieved by increasing the daily IVASP dosage to 100 mg/kg per day or more.

Effect of protein deprivation on insulin-like growth factor-binding proteins in rats.

The effect of protein deprivation on plasma concentration of insulin-like growth factor-binding proteins (IGFBP) was studied in rats. A significant decrease in the concentration of IGFBP of molecular weight (mass) approximately 40 kDa was observed in protein-deprived rats. There was no prominent effect of protein deprivation on the concentration of IGFBP with molecular weights of about 30 kDa or 22-24 kDa. The binding capacity to plasma IGFBP of exogenously-added 125I-labelled insulin-like growth factor-1 (125I-IGF-1) was also studied. IGFBP of molecular weight about 30 and 22-24 kDa (the native form of this protein is presumed to be 29 kDa) in protein-deprived rat plasma bound more 125I-IGF-1 than those in protein-fed rat plasma. This suggested that these IGFBP in protein-deprived rat plasma are relatively unsaturated by endogenous IGF-1. The response of IGFBP to protein deprivation which was elucidated in the present investigations add further evidence to our previous assumption that IGFBP play an important role in protein nutrition.

Formation of lignans (-)-secoisolariciresinol and (-)-matairesinol with Forsythia intermedia cell-free extracts.

In vivo labeling experiments of Forsythia intermedia plant tissue with [8-14C]- and [9,9-2H2,OC2H3]coniferyl alcohols revealed that the lignans, (-)-secoisolariciresinol and (-)-matairesinol, were derived from two coniferyl alcohol molecules; no evidence for the formation of the corresponding (+)-enantiomers was found. Administration of (+-)-[Ar-3H]secoisolariciresinols to excised shoots of F. intermedia resulted in a significant conversion into (-)-matairesinol; again, the (+)-antipode was not detected. Experiments using cell-free extracts of F. intermedia confirmed and extended these findings. In the presence of NAD(P)H and H2O2, the cell-free extracts catalyzed the formation of (-)-secoisolariciresinol, with either [8-14C]- or [9,9-2H2,OC2H3]coniferyl alcohols as substrates. The (+)-enantiomer was not formed. Finally, when either (-)-[Ar-3H] or (+-)-[Ar-2H]secoisolariciresinols were used as substrates, in the presence of NAD(P), only (-)- and not (+)-matairesinol formation occurred. The other antipode, (+)-secoisolariciresinol, did not serve as a substrate for the formation of either (+)- or (-)-matairesinol. Thus, in F. intermedia, the formation of the lignan, (-)-secoisolariciresinol, occurs under strict stereochemical control, in a reaction or reactions requiring NAD(P)H and H2O2 as cofactors. This stereoselectivity is retained in the subsequent conversion into (-)-matairesinol, since (+)-secoisolariciresinol is not a substrate. These are the first two enzymes to be discovered in lignan formation.