RESUMEN

Liverworts are a rich source of a diverse array of specialized metabolites, such as terpenoids and benzenoids, which are potentially useful for pharmaceutical or agrochemical applications, and also provide clues to elucidate the strategy by which liverworts adapt to the terrestrial environment. Liverworts, belonging to orders Marchantiales and Jungermanniales, possess oil bodies. In Marchantia polymorpha L., oil bodies are confined to scattered idioblastic oil body cells. It has been assumed that the specialized metabolites in M. polymorpha specifically accumulate in the oil bodies in oil body cells; however, no direct evidence was previously available for this specific accumulation. In this study, direct evidence was obtained using micromanipulation techniques coupled with MS analysis that demonstrated the specific accumulation of sesquiterpenoids and marchantin A in the oil body cells of M. polymorpha thalli. It was also observed that the number of oil body cells increased in thalli grown in low-mineral conditions. The amounts of sesquiterpenoids and marchantin A detected in crude extract prepared from the whole thallus were roughly proportional to the number of oil body cells found in a given volume of thallus, suggesting that oil body cell differentiation and sesquiterpenoid and marchantin A biosynthetic pathways are coordinated with each other.

Asunto(s)

RESUMEN

A droplet of an electroconductive solution was put on the sample plate of a matrix-assisted laser desorption/ionization-time of flight-mass spectroscope (MALDI-TOF-MS) and the outlet terminal of a capillary Electrophoresis (CE) capillary was put into this droplet in order to make an electro-connection and to apply high voltage between the metallic sample plate and the counter pole of the CE. This procedure was simple and gave much more stable interfacing than that of the electrospray method. Furthermore, the separated component was collected and concentrated in a droplet. By mixing each separated sample spot with the MALDI matrix on the sample plate, the spots were analyzed in separated sequences to make three-dimensional mass chromatograms, or applied to the enzyme digestion analysis for peptide sequencing.

Asunto(s)

RESUMEN

A high-performance capillary electrophoresis (HPCE) was successfully applied to the separation and quantitation of naturally occurring oleanene triterpenoidal saponins. The HPCE adapted to the separation of two pairs of disteriomeric saponins (1-2) or (3-4), obtained from Trifolium alexandrinum seeds, was based on capillary zone electrophoresis (CZE) in borate buffer with UV detection at 195 nm. An usual technique for isolation and group separation of saponins was developed as an appropriate purification step prior to determination of individual saponins by CZE. The separation parameters such as borate concentration, pH and applied voltage were varied in order to find the best compromise that complied with demands for high separation, short duration and sufficiently high detector response. The optimum running conditions were found to be 60 mM borate buffer, pH 10 and 12 kV. Under the alkaline borate electrolyte, no resolution was achieved for the saponins (1 and 3) or (2 and 4) in a single mixture, except when 20 mM beta-cyclodextrin was added to the running electrolyte. With the combined techniques of group separation, purification and CZE, a rapid and efficient method for the determination of naturally occurring diasteriomeric saponins is now available.

Asunto(s)

RESUMEN

The effects on humans of inhalation of optically active linalools were examined in terms of sensory tests and portable forehead surface electroencephalographic (IBVA-EEG) measurements in order to assess their odor distinctiveness by chiral isomers. (R)-(-)-Linalools with specific rotation of [alpha](D) = -15.1 degrees were isolated by repeated flash column chromatography from lavender oil, while (S)-(+)-linalools with [alpha](D) = +17.4 degrees and (RS)-(+/-)-linalools with [alpha](D) = 0 degrees and content of (R)-form 50.9% and (S)-form 49.1% were obtained from coriander oil and commercial linalool, respectively, by using the same method. With the use of an inhalator, each was administered to subjects both before and after 10 min of work. It was found that administration after work evoked different subjective impressions when compared with that before work depending on the configuration of the isomers and the type of work employed. For instance, inhalation of (R)-(-)-linalool after hearing environmental sounds not only produced a much more favorable impression in the sensory test but was also accompanied by a greater decrease in beta waves after work in comparison with that before work. This is in contrast to the case of mental work, which resulted in a tendency for agitation accompanied by an increase in beta waves. These findings led us to conclude that enantiomeric stereospecificity of linalool evoked different odor perception and responses not only with chiral dependence but also with task dependence. In addition, in comparing these sensory profiling features and IBVA-EEG tendencies between hearing environmental sound and mental work, a tendency was observed for (R)-(-)-linalool to coincide with (RS)-(+/-)-linalool but not with (S)-(+)-linalool.

Asunto(s)

RESUMEN

The stability of adriamycin (ADR), adriamycinol, adriamycinone (ADR-ONE) and daunomycin in the presence of alpha-, beta- and gamma-cyclodextrins (CDs) was studied using high-performance liquid chromatography. It was found that alpha-CD did not affect the degradation of tested compounds, beta-CD caused a little effect and gamma-CD resulted in pronounced stabilizing effect. The formation of complexes between ADR and ADR-ONE with CDs was monitored by fluorescence spectroscopy. The fluorescence spectrum of ADR-gamma-CD complex had an activation maximum at 460 nm, emission maximum at 555 nm and a shoulder at 585 nm. A similar finding was observed in case of alpha-CD. In case of beta-CD, the fluorescence intensity at 580 nm peak enhanced less than in case of gamma-CD. With ADR-ONE, alpha-CD did not cause any significant change compared with the spectrum of free molecule. On the other hand, it was noticed that, the fluorescence spectra of ADR-ONE with both beta- and gamma-CD were the same but showed a significant difference to the spectrum of free molecule, especially the molar fluorescence of the 585 nm emission peak.

RESUMEN

Mitotic index (MI), defined as the ratio (%) of the mitotic-phase-arrested cell number to the total cell number, is higher in the presence of colcemid than in its absence. MI is reduced by anti-tumor agents used in conjunction with colcemid. The ratio of MI, which is defined by the dose of colcemid to that of the supplement with etoposide as an anti-tumor agent and is designated MIR, was 10% at an exposure of 2 h, suggesting that etoposide blocks the cell cycle before 2 h of the mitotic phase. Thus, we established a convenient method to identify a target point in the cell cycle for anti-tumor agents using colcemid. RBL-2H3, a mammalian cell line, exhibited a large number of 2-fold swollen nuclear envelopes after exposure to etoposide. The G2-index, which is the ratio of the number of swollen nuclei to the total number of nuclei, was higher when the cells were incubated with etoposide than without it. In this study, we suggest that the measurement of the G2-index contributes to the screening of putative anti-tumor agents.

RESUMEN

The perceptional change of fragrance of essential oils is described in relation to type of work, i.e. mental work, physical work and hearing environmental (natural) sounds. The essential oils examined in this study were ylang ylang, orange, geranium, cypress, bergamot, spearmint and juniper. In evaluating change in perception of a given aroma, a sensory test was employed in which the perception of fragrance was assessed by 13 contrasting pairs of adjectives. Scores were recorded after inhaling a fragrance before and after each type of work, and the statistical significance of the change of score for 13 impression descriptors was examined by Student's t-test for each type of work. It was confirmed that inhalation of essential oil caused a different subjective perception of fragrance depending on the type of work. For example, inhalation of cypress after physical work produced a much more favorable impression than before work, in contrast to orange, which produced an unfavorable impression after physical work when compared with that before work. For mental work, inhalation of juniper seemed to create a favorable impression after work, whereas geranium and orange both produced an unfavorable impression then. From these studies, together with those conducted previously with lavender, rosemary, linalool, peppermint, marjoram, cardamom, sandalwood, basil and lime, we thus concluded that the sensory test described here might serve not only as a screening test for efficacy of aroma but also as a categorized table for aroma samples which can act as a reference to each other.

Asunto(s)

RESUMEN

A high-performance liquid chromatographic system, combining solid-phase extraction and automated precolumn derivatization is described for the routine determination of methotrexate in human plasma. The sample extraction and elution onto the analytical column were performed automatically and concomitantly using the column-switching technique and a protein-coated precolumn. Cerium (IV) trihydroxyhydroperoxide (CTH) was introduced as a packed oxidant before the analytical column for the conversion of methotrexate into highly fluorescent products. The oxidative-cleavage of methotrexate occurs during the flow of 0.04 M phosphate buffer (pH 3.5) containing plasma sample through CTH column with a flow rate of 0.5 mL/min at 40 degrees C. The fluorescent products were transferred to the protein-coated precolumn, which was then flushed with the same buffer for clean-up and enrichment from plasma sample. The trapped substances were desorbed from the precolumn and eluted onto the ODS/TM analytical column by isocratical elution with a mobile phase containing 0.05 M phosphate buffer, pH 6.6 and acetonitrile (90-10, v/v) for subsequent separation. The fluorescent products were detected fluorimetrically at excitation and emission wavelengths of 367 and 463 nm, respectively. The complete analysis was achieved within 15 min per sample. The calibration graph was linear in the range of 50-500 ng/mL of methotrexate and there was no interference from endogenous components.

Asunto(s)

RESUMEN

The insulin content in mouse insulinoma MIN6 cells was determined using matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry (TOF/MS). A mass spectrum of cellular insulin was obtained with a cell burst of MIN6 by hypotonic water. Signal intensities of intracellular insulin were proportional to the number of MIN6 cells. The present method was applied to the determination of intracellular insulin content of MIN6 before and after glucose stimulation.

Asunto(s)

RESUMEN

Particles of HPLC resins are used for the trapping of secreted molecules from a single cell. The basic molecules, e.g., histamine, are secreted from a single RBL-2H3 cell by granule exocytosis and are trapped by cation-exchange HPLC resins outside the cell. Since quinacrine is concentrated into the exocytotic and acidic microgranules in RBL-2H3 cells, which are used as a model cell line of mast cells, we measured the change in the fluorescence intensity of the quinacrine released from the cells and that of molecules trapped on the resin using a videomicroscope. By measuring the increase in the fluorescence intensity of the resins, we can estimate the real time course of molecular secretion from a single cell.

Asunto(s)

RESUMEN

Conventional in vitro studies of pepsinogen secretion have measured secretion into the bulk medium and have demonstrated the critical role of Ca2+ in the process. The present study was undertaken to obtain further details of the process of secretion and its relation to Ca2+ changes over very short time periods. The relation between Ca2+ mobilization and exocytosis in an isolated individual peptic cell of the bullfrog was investigated by a method to measure both intracellular Ca2+ ([Ca2+]i), using a fluorescent Ca2+ indicator, fura 2, and exocytosis from single cells using a video microscope analyzing system. Bombesin (3.2 x 10(-7) M) and bethanechol (3.2 x 10(-4) M) caused a rapid increase in [Ca2+]i (initial peak) and a corresponding high frequency of initial exocytosis. After the initial peak, [Ca2+]i was maintained at a somewhat elevated level over the baseline (sustained phase), with a corresponding low frequency of exocytosis. Both the sustained phase of elevated [Ca2+]i and the related exocytosis were eliminated by the depletion of extracellular Ca2+. Low concentrations of bombesin (3.2 x 10(-10) M) and bethanechol (3.2 x 10(-7) M) caused sustained low-amplitude Ca2+ oscillations with correspondingly low frequencies but also caused sustained exocytosis. These data show that 1) cellular response differs between high and low concentrations of stimulus, 2) there is a close relation between [Ca2+]i and exocytosis, 3) exocytosis follows elevation of [Ca2+]i by 14-45 s (n = 6), and 4) there is a significant positive correlation between the peak [Ca2+]i and the number of exocytoses.

Asunto(s)

RESUMEN

Separation process in a liquid chromatographic column were visualized and analyzed by a developed chromato-videoscope. The migration aspects were evaluated with successively obtained densitograms. In reversed-phase chromatography, the band widths of each solute band were almost equal fore both weakly and strongly retained solutes when compared at the same column position (the same migration distance in the column). In the gradient elution mode, the position of the solute band showed that the migration velocity of the solute band changed gradually according to changes in solvent composition. The drug trapping process to BSA- coated ODS packings for direct injection of biological fluid was also observed. In the absence of BSA from the sample solution, the drug molecules were trapped in a narrow band. However, at higher BSA concentrations in the sample solution, a broader band shape was observed. This band broadening shows how the drug molecules were retained on the protein.

Asunto(s)

RESUMEN

Real-time analysis of molecular dynamics in living cells was studied by developed video-microscopes. Two new detective methods were reported, one is for analysis of ciliary movement and the other is the qualitative analysis of exocytosis of insulin-containing granules with a video-enhanced light/fluorescent microscope. For analysis of ciliary movement, glass beads were migrated in the flow. The migration speed parallel to the flow produced by ciliary beating was used as an index of the beating activity. When tracheal epithelium isolated from mouse was incubated with ambroxol, and expectorant known to activate ciliary beat frequency, the floating speeds of glass beads were changed with 1 min of incubation. The results suggest that the present method is useful not only for screening of expectorants but also for the study of molecular mechanisms underlying ciliary beat of tracheal epithelium. Visualization of the moment of the release of contents from insulin-containing granules was achieved using video-enhanced fluorescent microscopy in MIN6 cells of mouse insulinoma cell line. A fluorescent amino acridine dye, quinacrine, was found to be incorporated into low-pH secretory granules, including insulin, in the cells. The granules which incorporated quinacrine emitted a slightly blue-green fluorescence. Upon stimulation with glucose, release of the quinacrine fluorescence from granules were observed. The present method would be useful for quantitative analysis of secretion of insulin from MIN6 cells as well as pancreatic beta-cells.

Asunto(s)

RESUMEN

Exocytotic responses associated with phagocytosis were investigated in a single neutrophil with a special reference to their dynamic properties and their spatiotemporal relationships with ionic and chemical responses during phagocytosis. The real-time sequence of phagocytosis-exocytosis was directly visualized by video-enhanced contrast differential interference contrast (VEC-DIC) microscopy. The actual release of contents from such a granule was proven by examining a cell loaded with quinacrine with a dual imaging system that allowed us to observe DIC and fluorescence images simultaneously at a high magnification. During the process of phagosome formation in a neutrophil engulfing an opsonized zymosan, the exocytotic response was observed first in a granule located near the cell surface initially attached to the zymosan, and then in other granules sequentially along pseudopodia surrounding the zymosan. When the phagocytosis was induced in a medium containing luminol, a chemiluminescence due to active oxidants was detected exclusively in the region of phagosome, suggesting that exocytosis took place on the phagosomal membrane and not on the plasma membrane. Changes in cytosolic free calcium concentration ([Ca2+]i) were further measured using fura-2 under the dual imaging system. [Ca2+]i transients were more closely related to the extension of pseudopodia for engulfing zymosan and not directly to the exocytosis. These findings lead to a conclusion that exocytosis associated with phagocytosis is initiated by attachment of the cell membrane to the invading organism and mediated by local activation of the phagosomal membrane.

Asunto(s)

RESUMEN

A high-performance liquid chromatographic method involving on-line precolumn oxidative cleavage and fluorimetric detection was developed for the determination of methotrexate in plasma. Plasma samples were subjected to protein precipitation followed by solvent purification and then injection into the chromatographic system. Cerium (IV) trihydroxyhydroperoxide (CTH) was introduced as a packed oxidant before analytical column for the conversion of methotrexate into highly fluorescent 2,4-diaminopteridine derivatives. The oxidative cleavage of methotrexate occurs during the flow of 0.04 M phosphate buffer (pH 3.5) containing the drug through CTH column with a flow-rate of 0.2 mL/min at 40 degrees C. The separation was performed on a reversed-phase column (ODS/TM) using a mobile phase consisting of phosphate buffer (0.05 M, pH 6.6) and acetonitrile (90:10 v/v). The fluorescent products were monitored fluorimetrically at emission and excitation wavelengths of 463 and 367 nm, respectively. Validation of accuracy and precision were satisfactory for both within- and between-run assays. All coefficients of variance were less than 4% and mean relative errors were within 1.11% to 7.83%. The average recovery was found to be 93.74% to 98.11%. The proposed method is highly sensitive, specific and applicable to biological fluids.

Asunto(s)

RESUMEN

Streptomyces castaneoglobisporus HUT6202 expresses an enzyme, tyrosinase, responsible for the production of melanin-like pigments. The present study revealed that the tyrosinase synthesis by the microorganism was induced about 80-fold, when young cells cultured for 6 h were incubated with methionine (Met) to the mid-log phase of growth, in comparison to without this amino acid. The Met-induced tyrosinase synthesis was inhibited by the addition of rifampicin and chloramphenicol, suggesting that transcriptional and translational events are necessary for the induction. We found that the addition of Cu2+ to the culture medium brings forward the period of expression of Met-induced tyrosinase activity.

Asunto(s)

RESUMEN

Rat basophilic leukemia (RBL-2H3) cells, which exhibit Ca2+-dependent secretion of granules when stimulated with antigen or the Ca2+-ionophore A23187, were observed under a video-enhanced light/fluorescence microscope. Exocytotic events of individual granules were visualized in individual cells stimulated with antigen or A23187. Exocytosis of granules stimulated with A23187 showed two peaks in the time course. The earlier one was inhibited by selective inhibitors of protein kinase C (Ro31-8425, Ro31-8220, and chelerythrine) and the other was inhibited by an inhibitor of phosphatidate hydrolase, propranolol. Exocytosis by antigen stimulation, however, showed only one peak, which was inhibited by the selective inhibitors of protein kinase C, but not by propranolol. These results indicate that at least two distinct components of exocytosis exist in RBL-2H3 cells.

Asunto(s)

RESUMEN

Exocytosis of secretory granules, including histamine, in rat basophilic leukemia (RBL-2H3) cells, which exhibit Ca(2+)-dependent secretion of granules when stimulated with antigen or Ca(2+)-ionophore (A23187), was observed under a video-enhanced light microscope. Exocytotic events of individual granules including fusion, extrusion, and membrane retrieval were visualized in individual cells stimulated with antigen or A23187. Exocytosis of granules stimulated with A23187 showed two peaks in its time courses. The earlier one of the peaks was inhibited by wortmannin ( > 100 nM), as an inhibitor of myosin light chain kinase (MLCK), and the other was not. Exocytosis by antigen-stimulation, however, showed only one peak, which was inhibited by low concentration of wortmannin ( < 50 nM) as an inhibitor of phosphatidylinositol 3-kinase (PI3-kinase). These results indicate that quantitative analysis of exocytosis visualized by video-enhanced light microscope reveals two different pathways, through P13-kinase and MLCK, of inhibitory effects on exocytosis by wortmannin in RBL-2H3 cells.

Asunto(s)

RESUMEN

Streptomyces castaneoglobisporus HUT6202 overproduces a diffusible melanin-like pigment. An operon, designated mel, containing a gene that encodes tyrosinase, which is involved in the synthesis of melanin pigment, was cloned from the chromosomal DNA of the microorganism into the high-copy plasmid pAK114 and expressed in S. lividans. The tyrosinase activity of the transformed cells was at approximately a 110-fold higher level than that of the same host carrying the plasmid pIJ702, which has the same replication origin as pAK114 and carries the mel operon from S. antibioticus. The sequence analysis of the S. castaneoglobisporus mel operon revealed that an open-reading frame consisting of 378 base pairs(bp), designated ORF378, was found upstream of the tyrosinase gene (TYRC) consisting of 819 bp. In the present study, we constructed a chimeric mel operon consisting of ORF378 from S. castaneoglobisporus and the tyrosinase gene (TYRA) from S. antibioticus. The chimeric mel operon or the S. antibioticus mel operon, which consists of ORF438 and TYRA, expressed the tyrosinase activity in Escherichia coli intracellularly when located under the control of lacZ promoter, and the tyrosinase activity from the former was at a 30-fold higher level than that from the latter. This suggests that the gene contributing to the high expression of the tyrosinase activity in S. castaneoglobisporus is ORF378, rather than TYRC.

Asunto(s)

RESUMEN

Adriamycin, adriamycinol, adriamycinone and duanorubicin were simultaneously determined by the development of an on-line plasma clean-up system. A short protein-coated Lichrosorb, RP-8, RP-2, CN and muBondapak phenyl as well as ODS silica have been examined for their performance as pre-columns. The drugs and metabolites were separated from weakly retained plasma components through two steps; phosphate buffer saline, pH 7.4 and 15% acetonitrile in 0.1 M sodium dihydrogen phosphate, pH 3. The chromatographic conditions were: ODS/TM column, flow rate 1 ml/min, 35% acctonitrile in 0.1 M sodium dihydrogen phosphate (pH 3) containing 0.3% heptafluorobutyric acid as mobile phase. The detection was carried out using fluorescence monitor operated at an emission 555 nm and excitation 460 nm. Good resolution was obtained within 13 min. This method is reproducible for analysis of drugs and metabolites (99.3-100.1%, CV < 2%) in plasma.