Acid-catalyzed rearrangement of aryl-substituted homobenzoquinone epoxides.

The BF3-catalyzed reactions of diphenyl-substituted and endo-monophenyl-substituted homobenzoquinone epoxides proceeded through a regioselective oxirane ring opening followed by participation of a pi-aryl transannular cyclization to give the tricyclic diketo alcohols. The conformationally semirigid ethano-bridged diphenyl-substituted homologues also provided similar diketo alcohols and the subsequent ring-expanded cycloheptenedione (via a subsequent 1,2-acyl migration associated with cyclopropane ring opening), depending on the methyl-substitution pattern of the quinone frame. However, the exo-monophenyl-substituted and the rigid biphenyl-2,2'-diyl-substituted homobenzoquinone epoxides essentially remained unchanged.

Induction of senescence-like phenotypes by forced expression of hic-5, which encodes a novel LIM motif protein, in immortalized human fibroblasts.

The hic-5 gene encodes a novel protein with Zn finger-like (LIM) motifs, the expression of which increases during cellular senescence. The ectopic expression of hic-5 in nontumorigenic immortalized human fibroblasts, whose expression levels of hic-5 were significantly reduced in comparison with those of mortal cells, decreased colony-forming efficiency. Stable clones expressing high levels of hic-5 mRNA showed higher levels of mRNAs for several extracellular matrix-related proteins, along with the alteration of an alternative splicing as seen in senescent cells and decreased c-fos inducibility. Furthermore, these clones acquired a senescence-like phenotype, such as growth retardation; senescence-like morphology; and increased expression of Cip1/WAF1/sdi1 after 20 to 40 population doublings. On the other hand, antisense RNA expression of hic-5 in human normal diploid fibroblasts delayed the senescence process. HIC-5 was localized in nuclei and had affinity for DNA. Based on these observations, we speculated that HIC-5 affected the expression of senescence-related genes through interacting with DNA and thereby induced the senescence-like phenotypes. To our knowledge, hic-5 is the first single gene that could induce senescence-like phenotypes in a certain type of immortalized human cell and mediate the normal process of senescence.

The regulatory region and transcription factor required for the expression of rat and salmon pituitary hormone-encoding genes show cell-type and species specificity.

The promoter regions of the genes encoding the rat and chum salmon growth hormones (GH) and rat prolactin (PRL) were combined with a reporter gene and introduced into GH- and/or PRL-producing cells from rat. The rat GH and PRL promoters (pGH and pPRL, respectively) were most active in cells producing GH and PRL, respectively. The activity of the salmon pGH was much less than that of the rat pGH in rat GH-producing cells. The regulatory region required for cell-type-specific gene expression of pituitary hormones thus contains information, not only for cell-type specificity, but possibly for species specificity as well. A reporter plasmid containing the GH or somatolactin (SL) promoter and an effector plasmid having a gene encoding transcription factor Pit-1 (rat or salmon) were cotransfected into HeLa (human) or EPC (carp) cells. Rat and salmon Pit-1 were more active in HeLa and EPC cells, respectively, indicating that Pit-1 appears to interact species specifically with the transcription machinery.

Mechanism of caffeine modulation of the antitumor activity of adriamycin.

We examined the effects of a combination of adriamycin (ADR) and caffeine on DNA and protein biosynthesis and on the activities of DNA polymerase alpha and beta in normal and tumor tissue. The decrease in DNA and protein biosynthesis in tumor produced by caffeine combined with ADR were 2.5 and 2.4 times greater, respectively, compared with ADR alone. The combination of caffeine and ADR enhanced the decrease in DNA polymerases activities in the tumor which was induced by ADR, the decreases being 1.8 and 1.6 times greater, respectively, than that of ADR alone. In contrast, these ADR-induced changes in normal tissues were not enhanced by the combination with caffeine. The combination with caffeine had no effect on ADR concentration in normal tissues, but in the tumor, it increased the ADR concentration to 2.1 times that of ADR alone. In vitro, ADR efflux from Ehrlich ascites carcinoma cells was significantly inhibited by exposure to caffeine. These findings indicate that the effect of caffeine on ADR concentration in the cell plays an important role in the mechanism by which caffeine enhances ADR antitumor activity.

A mini-scale mass production and separation system for secretory heterologous proteins by perfusion culture of recombinant Pichia pastoris using a shaken ceramic membrane flask.

The perfusion culture technique using a shaken ceramic membrane flask (SCM flask) was applied to the production of a secretory heterologous protein. A recombinant methylotrophic yeast strain, Pichia pastoris, was cultured aerobically on a reciprocal shaker using an SCM flask. High-level production of human serum albumin (HSA) was attempted by increasing both the cell concentration and the expression level of the recombinant gene. In the two-stage culture method, the cell concentration was first raised to 17 g/l by feeding glycerol, after which the expression of HSA was induced by feeding methanol. However, the concentration of HSA in the effluent filtrate was as low as 0.15 g/l, while the cell concentration continued to increase. In contrast, HSA was effectively produced by feeding methanol from an early stage of the culture. In this case, the HSA concentration reached 0.24 and 0.46 g/l, respectively, using the growth-associated production method without and with aeration into the head space of the SCM flask. The results showed that supplying sufficient oxygen together with the growth-associated induction method are effective for obtaining high-level expression of the methanol-inducible recombinant gene of P. pastoris. An HSA concentration in the filtrate of 1.5 g/l was finally achieved when the cell concentration was increased to 53 g/l by supplying oxygen-enriched gas to the SCM flask. The yield and productivity of HSA reached 2.6-fold and 10-fold those obtained in an ordinary fed-batch culture using a shake flask, and these levels were readily achieved by continuous replenishment of the culture supernatant. The achievements made in this study should contribute to the development of a handy bioreactor system for mini-scale mass production of target proteins with separation at high purity.

Electrophoretic identification of garlic and garlic products.

We developed a rapid and simple method for identifying garlic and garlic products using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining. Samples were homogenized with 1% SDS or 6M urea and centrifuged. Supernatant containing garlic proteins was mixed with the same volume of loading buffer containing SDS and mercaptoethanol, heated in boiling water for 2 min, and applied to the wells of a ready-to-use polyacrylamide gradient gel (4-20%). Electrophoresis was performed 20 mA constant current for 2 h. The gel was stained with a silver staining kit and dried. Protein patterns of garlic and garlic products are different from those of other Allium plants such as onion, rakkyo, and caucas. The method was used to analyze samples of spice and garlic clove products. Absence of protein bands in garlic extract products suggests the products may contain less proteins and/or denatured proteins.

[Estimation of daily dietary intake of aluminum].

The daily dietary intake of aluminum was estimated through a total diet study from 1996 to 1998. In ten institutes, total diet study samples were prepared and their aluminum concentration was determined. The average daily intake of aluminum was 3.5 mg and the range was 1.8-8.4 mg. The validity of the analytical result was supported by analyses of certified reference materials.

Enantioselective Single-Crystal-to-Single-Crystal Photodimerization of Coumarin and Thiocoumarin in Inclusion Complexes with Chiral Host Compounds.

An intermolecular enantioselective photoreaction by a single-crystal-to-single-crystal transformation has been carried out for the first time, as is evident from X-ray structure analysis and X-ray powder diffractometric studies. This reaction, the dimerization of the title compound to cyclobutane derivative 1 (X=O, S), provides a good example for studying the mechanism of topochemical reactions in the crystal.

Caffeine modulates the antitumor activity and toxic side effects of adriamycin.

To improve both the survival and the response rate of patients, it is necessary to find modifiers which enhance the effects of known anticancer agents. In this study, we examined the effects of caffeine on the antitumor activity and side effects of antitumor agents, using CDF1 male mice. The combination of caffeine with adriamycin significantly enhanced the antitumor activity of adriamycin, by 1.5-fold and 1.9-fold in single- and multi-administration schedules, respectively, compared to the activity of adriamycin only. However, the combination of caffeine with cisplatin had no effect on the antitumor activity of cisplatin in either administration schedule. We examined two biochemical parameters, lipid peroxide level and glutathione peroxidase activity, as indices of adriamycin-induced side effects. The caffeine combination did not enhance the adriamycin-induced changes in these two parameters. Regarding the lethal effect of adriamycin, the caffeine combination had no effect in the single administration schedule, but in the multi-administration schedule, it tended to reduce this acute toxicity. These results suggest that the combination of caffeine with adriamycin can significantly increase the in vivo antitumor activity of this agent without increasing its side effects.

Synthesis of poly-L-glutamates containing 5-substituted uracil moieties.

The polymer reaction of cyclic derivatives of uracil and 5-bromouracil with polyacrylic and polymethacrylic acids, and poly-L-glutamic acid was studied in dimethylformamide solution. A similar reaction of cyclic derivatives of uridine with the polymeric acids was also made. Alkaline hydrolysis was further carried out on the polymers obtained.

Watersoluble synthetic nucleic acid analogs--polyethyleneimine derivatives containing nucleic acid bases--conformation and interactionwith nucleic acids.

Water soluble polyethyleneimine derivatives containing nucleic acid bases were found to interact with polynucleotides, DNA, RNA. The conformational change by formation of complex was observed by CD spectra and was discussed with the hypochromicity in UV spectra. The rates of interactions between nucleic acid bases in polymers were slow as shown by UV spectra, but the conformational changes of the polynucleotides were fast as shown by CD spectra. In the case of the uracil derivative (PEI-Hse-Ura), high value of CD spectra [theta] 2.80 = -8.0 x 10(-4) for the complex with DNA might be caused by psi type conformation of DNA.

Synthesis and interaction studies of oligo and polyamino acids derivatives containing nucleosides.

Nucleic acid analogs of L-lysine derivatives containing uridine and/or adenosine were synthesized. As dimer models, Lys(Urd)-Lys(Urd) and Lys(Urd)-Lys(Ado) were prepared by activated ester method. These dimers were found to form complex with Poly A, which was observed from hypochromicity of UV spectra. Furthermore poly-L-lysine derivative containing uridine was also prepared. The maximum hypochromicity value of this polymer model with PolyA was higher than that of dimer models.

Synthesis and interaction studies of watersoluble nucleic acid analogs containing serine as a spacer.

Watersoluble nucleic acid analogs containing L- or D-serine as a spacer were synthesized. Thymine was used as nucleic acid bases of these analogs. The base contents of these analogs were 93-94%. These analogs were found to form stable polymer complexes with Poly A or DNA by specific base-base interaction, which were observed from hypochromicity of UV spectra. In both cases, the maximum hypochromicity values of PEI-L-Ser-Thy were higher than that of PEI-D-Ser-Thy.

Liquid chromatographic determination of alliin in garlic and garlic products.

A liquid chromatographic (LC) method is proposed for the determination of alliin in garlic and garlic products. The method involves heating of the sample with water in a bath of boiling water followed by homogenization and centrifugation. Interfering components are eliminated by use of a Sep-Pak C18 cartridge as a clean up step before injection. The LC system with ultraviolet detection at 210 nm consists of a separation on a Zorbax TMS column and isocratic elution with water as a mobile phase. Fluorometric determination by ion-pairing chromatography with tetra-n-butylammonium bromide on a Nucleosil 5C18 column is also described. The overall recoveries of alliin added to garlic products were greater than 90%. Thin-layer chromatography and enzymatic degradation of alliin were performed for the confirmation of alliin detected in garlic products.

Simultaneous determination of nalidixic acid, oxolinic acid and piromidic acid in fish by high-performance liquid chromatography with fluorescence and UV detection.

A simple and rapid method for the simultaneous determination of nalidixic acid (NA), oxolinic acid (OXA) and piromidic acid (PMA) in cultured fish has been developed by high-performance liquid chromatography (HPLC). The drugs were extracted with 0.1% metaphosphoric acid-methanol (6:4), followed by a Sep-Pak C18 clean-up procedure. The HPLC separation was carried out on a Kaseisorb LC ODS 300-5 column (25 cm x 4.6 mm I.D.) using 5 mM phosphate buffer-acetonitrile (6:4) as a mobile phase. A fluorescence detector was used for NA and OXA at the excitation wavelength of 325 nm and the emission wavelength of 365 nm and an ultraviolet detector at 280 nm for PMA. The calibration graphs were rectilinear from 1 to 10 ng for OXA, from 2 to 20 ng for NA and PMA. The recoveries of NA, OXA and PMA added to fish were 81.5-85.3, 83.7-88.7 and 80.9-84.9%, respectively, with high accuracy. The limits of detection were 0.01 micrograms/g for each drug.

Immobilization of nucleoside on silica gel and specific separation of oligonucleotides.

Deoxyadenosine was immobilized on silica gel, in order to use as HPLC resins for selective separation of oligonucleotides. The longest retention time was observed for the complementary pd(T)4, and increased with decrease of temperature. This fact suggested that the main separation factor was the base pairing between complementary nucleic acid bases. These resins may be useful for separation of components of nucleic acids and polynucleotides as a specific separation system.

Synthesis and properties of oligolysine and oligoglutamic acid derivatives containing nucleosides.

Nucleic acid analogs of L-lysine derivatives containing uridine and/or adenosine were synthesized. As dimer models, Lys(Urd)-Lys(Urd) and Lys(Urd)-Lys(Ado) were prepared by activated ester method. These dimers were found to form complex with poly(A), which was observed from hypochromicity of UV spectra. Furthermore, glutamic acid derivatives containing uridine and adenosine were also prepared. Oligomerization of glutamic acid derivatives were studied by Merrifield's solid phase synthesis.

Structure and photodimerizations of 1-alkylthymines in single crystals.

1-Alkylthymine gave four kinds of crystals depending on the solvents used. Photodimerizations of 1-alkylthymine in single crystals were found to depend on the crystal structure. In inactive crystals, the terminal methyl group of alkyl chains approached to the double bond of thymine, and inhibited rotation of thymine bases during photodimerization.