Deodorizing effects of tea catechins on amines and ammonia

Deodorizing effects of tea catechins on amines were examined under alkaline conditions to eliminate the neutralization reaction. They showed deodorizing activity on ethylamine, but none on dimethylamine or trimethylamine. Deodorizing activity on ethylamine was found to be in the order of (-)-epigallocatechin gallate > gallic acid > (-)-epigallocatechin (EGC) > (-)-epicatechin gallate > ethyl gallate > (+)-catechin = (-)-epicatechin. Further, reaction products of EGC with methylamine, ethylamine, and ammonia were detected by HPLC, indicating that a deodorizing reaction other than neutralization occurs. From structural analysis of the reaction product with the methylamine isolated as a peracetylated derivative, the product was presumed to be methylamine substituted EGC, in which the hydroxyl group of EGC at the 4' position is replaced by the methylamino group. The same replacement reaction took place in the case of ethylamine and ammonia.     

Changes in flower coloration and sepal anthocyanins of Cyanic delphinium cultivars during flowering

The changes in flower color related to sepal pigmentation of cyanic Delphinium cultivars were investigated during anthesis. The sepal hues of the purple and blue flowered varieties observed on the initial day of unfurling had changed with a decrease in hue angle three days after anthesis. In both the purple and blue cultivars, violdelphin (3) was the major component on day one of anthesis, and the chromaticity improved with increasing sepal concentrations of violdelphin (3) and cyanodelphin (4) after three days of unfurling. The flower hue was dominated by the constitution of acylated anthocyanins, and the chromaticity was ordered by the sepal concentration. The biosynthesis of cyanodelphin (4) from violdelphin (3) was postulated since an increase in the sepal concentration of cyanodelphin (4) was accompanied by a decrease in violdelphin (3). Acylation of the anthocyanins was initiated by an increase in the respective possible precursors, tulipanin (2) and violdelphin (3), to subsequently synthesize violdelphin (3) and cyanodelphin (4) during flowering.     

Examination of morphological changes in the first formed protoxylem in Arabidopsis seedlings

We examined morphological changes in the first-formed protoxylem vessels in Arabidopsis seedlings. Between 2.5 and 8 days after imbibition, mean hypocotyl and root length increased 1.52 and 23.3 times, respectively. In the 2.5-day-old seedlings, two continuous protoxylem vessels were present in the hypocotyl-root axis. In the 8-day-old upper hypocotyls, six protoxylem vessels were observed, and in the lower hypocotyls, four protoxylem vessels and one or two metaxylem vessels were observed. In the 8-day-old roots, there were two protoxylem vessels and two or three metaxylem vessels. Two protoxylem vessels in the hypocotyls connected to two metaxylem vessels in the roots of 8-day-old seedlings. At the 0.3-mm part below the hypocotyl-root boundary, the mean intervals of neighboring annular secondary wall thickenings in protoxylem vessels in 8-day-old roots were 12.9% larger than those in 2.5-day-old roots. In more apical parts of 8-day-old roots, the mean intervals fluctuated between 1.71 and 2.29 microm. In 8-day-old seedlings, metaxylem vessels were formed between 0.4 mm above the hypocotyl-root boundary and 17 mm below the boundary. The intervals in these regions were not extended so much as protoxylem vessels were collapsed. The first-formed protoxylem vessels presumably retain their water-conductive function after metaxylem formation.     

Frameshifts and deletions during in vitro translesion synthesis past Pt-DNA adducts by DNA polymerases beta and eta

DNA polymerases beta (pol beta ) and eta (pol eta ) are the only two eukaryotic polymerases known to efficiently bypass cisplatin and oxaliplatin adducts in vitro. Frameshift errors are an important aspect of mutagenesis. We have compared the types of frameshifts that occur during translesion synthesis past cisplatin and oxaliplatin adducts in vitro by pol beta and pol eta on a template containing multiple runs of nucleotides flanking a single platinum-GG adduct. Translesion synthesis past platinum adducts by pol beta resulted in approximately 50% replication products containing single-base deletions. For both adducts the majority of -1 frameshifts occurred in a TTT sequence 3-5 bp upstream of the DNA lesion. For pol eta, all of the bypass products for both cisplatin and oxaliplatin adducts contained -1 frameshifts in the upstream TTT sequence and most of the products of replication on oxaliplatin-damaged templates had multiple replication errors, both frameshifts and misinsertions. In addition, on platinated templates both polymerases generated replication products 4-8 bp shorter than the full-length products. The majority of short cisplatin-induced products contained an internal deletion which included the adduct. In contrast, the majority of oxaliplatin-induced short products contained a 3' terminal deletion. The implications of these in vitro results for in vivo mutagenesis are discussed.     

Studies on scavenger receptor inhibitors. Part 1: synthesis and structure-activity relationships of novel derivatives of sulfatides

Scavenger receptors have been proven to be implicated in the formation of atherosclerotic lesions. A series of novel derivatives of sulfatides were synthesized, and their inhibitory activities against incorporation of DiI-acetyl-LDL into macrophages were evaluated in order to clarify the structure-activity relationships of sulfatides as a scavenger receptor inhibitor and find out novel inhibitors with synthetic easiness. The chemical modification of the substructures of sulfatides led to the establishment of the following structure-activity relationships; (1) the ceramide moiety can be replaced with another structure bearing two long chains, (2) the galactose moiety can be replaced with another structure or be deleted without a large decrease in the inhibitory activity, (3) the sulfate moiety was crucial, and it was the most preferable functional group for a potent inhibitory activity. The inhibitory activity of (S)-2-octadecanoylamino-2-tetradecylcarbamoyl)ethyl sulfate sodium salt (3a) against incorporation of DiI-acetyl-LDL into macrophages was proven to be based on the inhibition against the binding of acetyl-LDL to the surface of macrophages. We discovered novel scavenger receptor inhibitors with synthetic easiness, such as (S)-2-octadecanoylamino-2-(tetradecylcarbamoyl)ethyl sulfate sodium salt (3a) and 2-octadecanoylamino-1-(octadecanoylaminomethyl)ethyl sulfate sodium salt (13q).     

Small aggregates of platelets can be detected sensitively by a flow cytometer equipped with an imaging device: mechanisms of epinephrine-induced aggregation and antiplatelet effects of beraprost

BACKGROUND: Although cross-talks between platelets and other blood cells are important in vivo, laboratory platelet aggregation tests have been performed mainly with the use of platelet-rich plasma (PRP) as samples. Methods that enable an efficient and sensitive detection of platelet aggregates in whole blood are being developed. METHODS: A flow cytometer equipped with an imaging device, the flow imaging cytometer 2 (FIC2), was used to detect platelet aggregates in whole blood. RESULTS: The FIC2 provides a resolution that is high enough to differentiate platelet aggregates from single platelets or other blood cells. Epinephrine elicited platelet aggregate formation in hirudin plus argatroban-treated whole blood, but not in PRP. The reconstitution study revealed that a small amount of adenosine diphosphate (ADP) from erythrocytes may play an important role in epinephrine-induced platelet aggregation (in whole blood), through mediation of P2Y1 receptors. When the inhibitory effect of beraprost, an antiplatelet agent, on platelet aggregation was assessed, analysis of whole blood samples with FIC2 proved to be the most sensitive among the methods available. CONCLUSIONS: FIC2 is a promising device for detection of platelet aggregates in whole blood, with wide basic and clinical applications.     

A novel apoptosis research method with imaging-combined flow cytometer and HITC or IR-125 staining

The most commonly used methods for apoptotic research include terminal transferase-mediated dUTP nick end-labeling, annexin V testing of phosphatidylserine translocation from the inner leaflet to the outer plasma membrane by flow cytometry, DNA electrophoresis, and cell morphology. These methods provide apoptotic information from different aspects. To find a new way in apoptosis research and potential clinical application, we recently developed a novel method with an imaging-combined flow cytometer (IFC) and an innovative cell staining process by using 2-[7-(1,3-dihydro-1,3,3-trimethyl-2H-indol-2-ylidene)-1,3,5-heptatrienyl]-1,3,3-trimethyl-3H-indolium iodide (HITC) and 2-[7-[1,3-dihydro-1,1-dimethyl-3-(4-sulfobutyl)-2H-benz[e]indol-2-ylidene]-1,3,5-heptatrienyl]-1,1-dimethyl-3-(4-sulfobutyl)-1H-benz[e]indolium hydroxide, inner salt, sodium salt (IR-125). The IFC used in the research is a new generation of cytometry designed for simultaneous observations of cell populations and images. This is possible because the IFC is equipped with dual laser beams, one argon and one infrared. A promyelocytic leukemia cell line, HL-60, was used in the research. The cells were stained with our newly developed HITC or IR-125 staining method and a traditional nucleic acid dye, propidium iodide. The cells stained with HITC or IR-125 appeared completely dark in the IFC image window before washing. Phosphate buffered saline wash did not change the cell appearance. A wash with 50% methanol caused the cells to have a clear cell image with bright nuclei on the IFC. To obtain apoptotic cells, we treated the HL-60 cells with 0.15 microM of camptothecin (CAM), a topoisomerase I inhibitor and experimental apoptosis inducer, for 4 h. The control showed larger round cells with bright nuclei and one to three dark nucleoli. The CAM-induced apoptotic cells were smaller, with fragmented and condensed nuclei on the IFC. These appearances were identical to the cell morphology of with light and electron microscopy. We used other methods including FACScan and DNA electrophoresis to confirm the apoptotic changes after CAM treatment and compared them with the IFC method. In addition, we found that the novel method with the IFC and HITC or IR-125 staining can show not only cell apoptotic changes but also peripheral blood cell populations and images simultaneously. This study suggests many potential applications of the IFC and this novel staining method in other cellular biological researches and clinical assays.     

Anti-bovine rhodopsin monoclonal antibody recognizing light-dependent structural change

The antigenic structure of the bovine rhodopsin molecule was investigated by using a bovine rhodopsin-specific monoclonal antibody designated Rh 29. Competition assay with sealed intact disks and broken disks indicated that the antibody-binding region was localized in the intradiscal surface. An antigenic peptide obtained by a cyanogene bromide cleavage of rhodopsin was purified and determined as residues 2-39 in the amino acid sequence. Further analysis suggested that the antigenic determinant included at least residues 21-25. These results were consistent with the structural model for membrane topology of rhodopsin. The antigenicity of the rhodopsin was compared among several states. The antibody bound to both ammonyx LO-solubilized unbleached and bleached rhodopsin. In contrast, upon membrane-embedded rhodopsin, unbleached one was 100-times less antigenic than bleached one. The results suggested that the segment around the determinant of membrane-embedded rhodopsin should undergo a structural change upon absorption of light. Rh 29 detected a band corresponding to bovine, porcine and octopus opsins in immunoblotting. Protein blot of crayfish rhabdome did not show any reactive band. These bands except for crayfish reacted with concanavalin A as well. The N-terminal structure may, therefore, conserved between mammal and erthropoda and diverge between them and cepharopoda.     

The XPC-HR23B complex displays high affinity and specificity for damaged DNA in a true-equilibrium fluorescence assay

The XPC-HR23B complex is a prime candidate for the initial damage recognition step during global genome nucleotide excision repair. A specific interaction between the XPC-HR23B complex and various types of damaged DNA substrates has been demonstrated in recent work by electrophoretic mobility shift assays or immunoprecipitation. Although these studies allowed the estimation of relative binding affinities for the different types of lesions, the presence of large amounts of competitor DNA or the need for glutaraldehyde fixation prevented the quantification of equilibrium constants. We have performed a quantitative study on the binding of XPC to damaged DNA using fluorescence anisotropy measurements. The XPC-HR23B complex binds with high affinity (K(D) approximately 1-3 nM) to fluorescent 36 bp DNA fragments containing a single cisplatin 1,3-intrastrand adduct or a six-nucleotide mispaired region. From stoichiometric titration experiments, it is concluded that approximately 70% of the XPC-HR23B preparation is active in DNA binding. Binding experiments employing fluorescent probes with a single defined photoproduct reveal a 30-fold preference of XPC for 6,4-photoproducts as compared to a cyclobutane dimer. Competition experiments with undamaged and damaged plasmid DNA indicate that the XPC-HR23B complex discriminates between damaged and undamaged sites with high specificity. The specificity factor is between 100 and 3000, depending on the number of nonspecific sites considered in the calculations. Upon addition of XPA to the XPC binding reaction mixtures, it was not possible to detect cooperative ternary complex formation on the platinated 36 bp probe.     

A role for DNA polymerase beta in mutagenic UV lesion bypass

We report here that DNA polymerase beta (pol beta), the base excision repair polymerase, is highly expressed in human melanoma tissues, known to be associated with UV radiation exposure. To investigate the potential role of pol beta in UV-induced genetic instability, we analyzed the cellular and molecular effects of excess pol beta. We firstly demonstrated that mammalian cells overexpressing pol beta are resistant and hypermutagenic after UV irradiation and that replicative extracts from these cells are able to catalyze complete translesion replication of a thymine-thymine cyclobutane pyrimidine dimer (CPD). By using in vitro primer extension reactions with purified pol beta, we showed that CPD as well as, to a lesser extent, the thymine-thymine pyrimidine-pyrimidone (6-4) photoproduct, were bypassed. pol beta mostly incorporates the correct dATP opposite the 3'-terminus of both CPD and the (6-4) photoproduct but can also misinsert dCTP at a frequency of 32 and 26%, respectively. In the case of CPD, efficient and error-prone extension of the correct dATP was found. These data support a biological role of pol beta in UV lesion bypass and suggest that deregulated pol beta may enhance UV-induced genetic instability.