Cell cycle synchronization of tobacco BY-2 cells

Synchronization is a powerful technique for understanding cell cycle events. Here, we describe the procedure for synchronizing tobacco bright yellow 2 (BY-2) cell line, with which an exceptionally high level of synchrony can be achieved. It basically relies on an "arrest-and-release" strategy using aphidicolin, an inhibitor of DNA replication, and propyzamide, a plant-microtubule disruptant. In a single-step process using aphidicolin alone, a cell population with about 70% of the cells at mitosis can be achieved, whereas by a two-step method using the two inhibitors sequentially, the level of synchrony can reach over 90%. The method of choice depends not only on the peak mitotic cell proportion but also on the cell cycle stage that is targeted for analysis. Both procedures take about 1.5 days, and cell cycle progression can be observed from the S phase to the next G1 phase at about 12 h after a 24 h-period treatment with aphidicolin.     

4-vinyl-substituted pyrimidine nucleosides exhibit the efficient and selective formation of interstrand cross-links with RNA and duplex DNA

The formation of interstrand cross-links in nucleic acids can have a strong impact on biological function of nucleic acids; therefore, many cross-linking agents have been developed for biological applications. Despite numerous studies, there remains a need for cross-linking agents that exhibit both efficiency and selectivity. In this study, a 4-vinyl-substituted analog of thymidine (T-vinyl derivative) was designed as a new cross-linking agent, in which the vinyl group is oriented towards the Watson–Crick face to react with the amino group of an adenine base. The interstrand cross-link formed rapidly and selectively with a uridine on the RNA substrate at the site opposite to the T-vinyl derivative. A detailed analysis of cross-link formation while varying the flanking bases of the RNA substrates indicated that interstrand cross-link formation is preferential for the adenine base on the 5′-side of the opposing uridine. In the absence of a 5′-adenine, a uridine at the opposite position underwent cross-linking. The oligodeoxynucleotides probe incorporating the T-vinyl derivative efficiently formed interstrand cross-links in parallel-type triplex DNA with high selectivity for dA in the homopurine strand. The efficiency and selectivity of the T-vinyl derivative illustrate its potential use as a unique tool in biological and materials research.     

Sphingosine 1-phosphate regulates the egress of IgA plasmablasts from Peyer's patches for intestinal IgA responses

It is well established that Peyer's patches (PPs) are sites for the differentiation of IgA plasma cell precursors, but molecular and cellular mechanisms in their trafficking remain to be elucidated. In this study, we show that alterations in type 1 sphingosine 1-phosphate (S1P) receptor expression during B cell differentiation in the PPs control the emigration of IgA plasma cell precursors. Type 1 S1P receptor expression decreased during the differentiation of IgM(+)B220(+) B cells to IgA(+)B220(+) B cells, but recovered on IgA(+)B220(-) plasmablasts for their emigration from the PPs. Thus, IgA(+)B220(-) plasmablasts migrated in response to S1P in vitro. Additionally, IgA(+) plasmablasts selectively accumulated in lymphatic regions of PPs when S1P-mediated signaling was disrupted by FTY720 treatment. This accumulation of IgA(+) plasmablasts in the PPs led to their reduction in the intestinal lamina propria and simultaneous impairment of Ag-specific intestinal IgA production against orally administered Ag. These findings suggest that S1P regulates the retention and emigration of PP B cells and plays key roles in the induction of intestinal IgA production.     

Tetra-acylated cyanidin 3-sophoroside-5-glucosides from the flowers of Iberis umbellata L. (Cruciferae)

The structures of 11 acylated cyanidin 3-sophoroside-5-glucosides (pigments 1-11), isolated from the flowers of Iberis umbellata cultivars (Cruciferae), were elucidated by chemical and spectroscopic methods. Pigments 1-11 were acylated with malonic acid, p-coumaric acid, ferulic acid, sinapic acid and/or glucosylhydroxycinnamic acids.Pigments 1-11 were classified into four groups by the substitution patterns of the linear acylated residues at the 3-position of the cyanidin. In the first group, pigments 1-3 were determined to be cyanidin 3-O-[2-O-(2-O-(acyl)-β-glucopyranosyl)-6-O-(trans-p-coumaroyl)-β-glucopyranoside]-5-O-[6-O-(malonyl)-β-glucopyranoside], in which the acyl moiety varied with none for pigment 1, ferulic acid for pigment 2 and sinapic acid for pigment 3. In the second one, pigments 4-6 were cyanidin 3-O-[2-O-(2-O-(acyl)-β-glucopyranosyl)-6-O-(4-O-(β-glucopyranosyl)-trans-p-coumaroyl)-β-glucopyranoside]-5-O-[6-O-(malonyl)-β-glucopyranoside], in which the acyl moiety varied with none for pigment 4, ferulic acid for pigment 5 and sinapic acid for pigment 6. In the third one, pigments 7-9 were cyanidin 3-O-[2-O-(2-O-(acyl)-β-glucopyranosyl)-6-O-(4-O-(6-O-(trans-feruloyl)-β-glucopyranosyl)-trans-p-coumaroyl)-β-glucopyranoside]-5-O-[6-O-(malonyl)-β-glucopyranoside], in which the acyl moiety varied with none for pigment 7, ferulic acid for pigment 8, and sinapic acid for pigment 9. In the last one, pigments 10 and 11 were cyanidin 3-O-[2-O-(2-O-(acyl)-β-glucopyranosyl)-6-O-(4-O-(6-O-(4-O-(β-glucopyranosyl)-trans-feruloyl)-β-glucopyranosyl)-trans-p-coumaroyl)-β-glucopyranoside]-5-O-[6-O-(malonyl)-β-glucopyranoside], in which acyl moieties were none for pigment 10 and ferulic acid for pigment 11.The distribution of these pigments was examined in the flowers of four cultivars of I. umbellata by HPLC analysis. Pigment 1 acylated with one molecule of p-coumaric acid was dominantly observed in purple-violet cultivars. On the other hand, pigments (9 and 11) acylated with three molecules of hydroxycinnamic acids were observed in lilac (purple-violet) cultivars as major anthocyanins. The bluing effect and stability on these anthocyanin colors were discussed in relation to the molecular number of hydroxycinnamic acids in these anthocyanin molecules.     

Application of liquid-based preparation to fine needle aspiration cytology in breast cancer

OBJECTIVE: To examine the performance of liquid-based cytology (LBC) in breast cytology to confirm the diagnosis of carcinoma. STUDY DESIGN: Using cell clusters directly scratched from surgically removed tumor masses, we examined the immunocytochemistry, molecular biology and cytomorphology of the specimens. RESULTS: LBC was very useful for gene analysis and evaluating the immunocytochemistry. The cytologic features of LBC were slightly different from those ofa conventional aspiration cytology smear. CONCLUSION: LBC is a promising method for improving the standardization ofpreparations in breast cytology, although care should be taken to account for its characteristic cytologic features. The quantitative analysis of HER-2 mRNA correlated with the results of immunohistochemistry.     

An Inducible Copper-Thiolate Complex in the Fern, Athyrium yokoscense: Involvement in Copper-Tolerance of the Fern

Two peaks of Cu-containing material were isolated from the solublecytoplasmic fraction of roots of Athyrium yokoscense, a fernwith a tolerance for heavy metals, by gel filtration on Bio-GelP-30 and Sephadex G-25. Peak I (apparent molecular weight 9.5kDa) was only observed in the case of ferns growing on Cu-contaminatedsoil. Peak II (apparent molecular weight 2 kDa) was found inextracts of ferns from both Cu-contaminated and uncontaminatedsoil. Peak I was induced in the fern collected from an uncontaminatedarea by cultivation in Cu-contaminated soil for 14 months. Theinduced Cu-binding complex present in peak I, therefore, maybe involved in mechanisms of Cu-tolerance in the fern. The complexwas purified by column chromatography on DEAE-cellulofine andsubsequent high-performance liquid chromatography. The purifiedcomplex contained a high percentage (26.8%) of cysteine, typicalof metal-binding peptides from plants. (Received June 1, 1988; Accepted September 12, 1988)     

Critical function of the Ras-associating domain as a primary Ras-binding site for regulation of Saccharomyces cerevisiae adenylyl cyclase.

Mammalian candidate effectors of the small GTPase Ras, such as RalGDS, afadin/AF-6, Rin1, and phospholipase Cepsilon, have been shown to share structurally conserved modules termed Ras-associating (RA) domains at their Ras-binding sites. The Ras-binding domains of Raf-1 and phosphoinositide 3-kinase gamma (other Ras effectors) also share a similar tertiary structure with the RA domains. On the other hand, the primary Ras-binding site of Saccharomyces cerevisiae adenylyl cyclase, the best characterized Ras effector, has been mapped by mutational studies to the leucine-rich repeats (LRR) domain (amino acids 674-1300), whose structure apparently bears no resemblance to the RA domains. By a computer algorithm-based search we have unexpectedly found an RA domain in the N-terminal 81 amino acid residues (676) of the LRR domain. The purified RA-domain polypeptide exhibits an ability to bind directly to Ras in a GTP-dependent manner and to competitively inhibit Ras-dependent activation of adenylyl cyclase in vitro, with an affinity comparable with that of the whole LRR domain. The specificity of binding of the RA domain to various Ras effector region mutants is indistinguishable from that of the full-length adenylyl cyclase. The activated RAS2 (RAS2(Val-19))-dependent heat shock sensitivity of yeast cells is suppressed by overexpression of the RA domain polypeptide. Further, mutations of the RA domain abolish its Ras binding activity, and adenylyl cyclase molecules carrying these mutations are rendered unactivatable by Ras in vitro. This RA domain bears highest similarity to the Ras-binding domain of Raf-1 based on comparison of its primary and predicted secondary structures with those of other Ras effectors. These results indicate that the RA domain is a primary Ras-binding site for activation of adenylyl cyclase, implicating RA domains as universal modules for interaction of effectors with Ras, conserved from yeast to mammals.     

Expansion of triplex recognition codes by the use of novel bicyclic nucleoside derivatives (WNA)

Recently, we have developed new base analogs (WNA) and demonstrated that WNA-[see text];T with thymine and WNA-[see text];C with cytosine stabilize n on-natural antiparallel triplexes with a TA or CG interrupting site, respectively. However, limitations in recognizable sequences with the WNA-containing TFO were also found. The objective of this study is to search better WNA analogs for expansion of triplex recognition codes to general duplex sequences. In this study, we designed new WNA analogs by systematic modification of the aromatic part and the recognition part. The new WNA analogs with the benzene ring substituted with bromide or cyanide have determined for selective stabilization of triplexes at a TA interrupting site, and general formation of triplexes having a TA interrupting site has been achieved.     

The blue anthocyanin pigments from the blue flowers of Heliophila coronopifolia L. (Brassicaceae)

Six acylated delphinidin glycosides (pigments 1-6) and one acylated kaempferol glycoside (pigment 9) were isolated from the blue flowers of cape stock (Heliophila coronopifolia) in Brassicaceae along with two known acylated cyanidin glycosides (pigments 7 and 8). Pigments 1-8, based on 3-sambubioside-5-glucosides of delphinidin and cyanidin, were acylated with hydroxycinnamic acids at 3-glycosyl residues of anthocyanidins. Using spectroscopic and chemical methods, the structures of pigments 1, 2, 5, and 6 were determined to be: delphinidin 3-O-[2-O-(β-xylopyranosyl)-6-O-(acyl)-β-glucopyranoside]-5-O-[6-O-(malonyl)-β-glucopyranoside], in which acyl moieties were, respectively, cis-p-coumaric acid for pigment 1, trans-caffeic acid for pigment 2, trans-p-coumaric acid for pigment 5 (a main pigment) and trans-ferulic acid for pigment 6, respectively. Moreover, the structure of pigments 3 and 4 were elucidated, respectively, as a demalonyl pigment 5 and a demalonyl pigment 6. Two known anthocyanins (pigments 7 and 8) were identified to be cyanidin 3-(6-p-coumaroyl-sambubioside)-5-(6-malonyl-glucoside) for pigment 7 and cyanidin 3-(6-feruloyl-sambubioside)-5-(6-malonyl-glucoside) for pigment 8 as minor anthocyanin pigments. A flavonol pigment (pigment 9) was isolated from its flowers and determined to be kaempferol 3-O-[6-O-(trans-feruloyl)-β-glucopyranoside]-7-O-cellobioside-4′-O-glucopyranoside as the main flavonol pigment.On the visible absorption spectral curve of the fresh blue petals of this plant and its petal pressed juice in the pH 5.0 buffer solution, three characteristic absorption maxima were observed at 546, 583 and 635 nm. However, the absorption curve of pigment 5 (a main anthocyanin in its flower) exhibited only one maximum at 569 nm in the pH 5.0 buffer solution, and violet color. The color of pigment 5 was observed to be very unstable in the pH 5.0 solution and soon decayed. In the pH 5.0 solution, the violet color of pigment 5 was restored as pure blue color by addition of pigment 9 (a main flavonol in this flower) like its fresh flower, and its blue solution exhibited the same three maxima at 546, 583 and 635 nm. On the other hand, the violet color of pigment 5 in the pH 5.0 buffer solution was not restored as pure blue color by addition of deacyl pigment 9 or rutin (a typical flower copigment). It is particularly interesting that, a blue anthocyanin-flavonol complex was extracted from the blue flowers of this plant with H₂O or 5% HOAc solution as a dark blue powder. This complex exhibited the same absorption maxima at 546, 583 and 635 nm in the pH 5.0 buffer solution. Analysis of FAB mass measurement established that this blue anthocyanin-flavonol complex was composed of one molecule each of pigment 5 and pigment 9, exhibiting a molecular ion [M+1] ⁺ at 2102 m/z (C₉₃H₁₀₅O₅₅ calc. 2101.542). However, this blue complex is extremely unstable in acid solution. It really dissociates into pigment 5 and pigment 9.