Synthetic nucleosides and nucleotides. 43. Inhibition of vertebrate telomerases by carbocyclic oxetanocin g (C.OXT-G) triphosphate analogues and influence of C.OXT-G treatment on telomere length in human HL60 cells.

Telomerase, responsible for telomere synthesis, is expressed in approximately 90% of human tumor cells but seldom in normal somatic cells. In this study, inhibition by carbocyclic oxetanocin G triphosphate (C. OXT-GTP) and its analogues was investigated in order to clarify the susceptibility of telomerase to various nucleotide analogues.

Delivery of exogenous tetrahydrobiopterin (BH4) to cells of target organs: role of salvage pathway and uptake of its precursor in effective elevation of tissue BH4.

Cells in target organs such as liver do not generally incorporate tetrahydrobiopterin (BH4) in its fully reduced form. Instead, they transiently take up BH4 from the extracellular fluid, instantaneously oxidize it and then expel virtually all of it. However, a small but stable accumulation of BH4 was observed after BH4 administration to the cell cultures.

Distinctive iron requirement of tryptophan 5-monooxygenase: TPH1 requires dissociable ferrous iron.

A peripheral type of tryptophan 5-monooxygenase (EC 1.14.16.

Cellular accumulation of tetrahydrobiopterin following its administration is mediated by two different processes; direct uptake and indirect uptake mediated by a methotrexate-sensitive process.

In our previous study on tetrahydrobiopterin (BH4) accumulation in organs of mice administered with 6RBH4, it was demonstrated that the intestinal mucosa was able to take up BH4 directly but that the liver could accomplish this only indirectly via a pathway involving the dihydrofolate reductase reaction. This observation was largely based on the fact that BH4 deposition in the liver was completely inhibited by prior treatment with methotrexate whereas deposition in the intestinal mucosa was only partially inhibited. To investigate the distinctive features of BH4 uptake in these organs, Caco-2 of intestinal epithelial origin and isolated hepatocytes were analyzed for cellular BH4 uptake in vitro.

Accumulated BH4 in mouse liver caused by administration of either 6R- or 6SBH4 consisted solely of the 6R-diastereomer: evidence of oxidation to BH2 and enzymic reduction.

Mice were given (i.p.) L-erythro-(6S)-tetrahydrobiopterin (6SBH4) or 6RBH4 and the increase in liver BH4 in both groups was almost the same.

Protein splicing: its discovery and structural insight into novel chemical mechanisms.

Protein splicing is a posttranslational cellular process, in which an intervening protein sequence (intein) is self-catalytically excised out from a nascent protein precursor and the two flanking sequences (N- and C-exteins) are ligated to produce two mature enzymes. This unique reaction was first discovered from studies of the structure and expression of the VMA1 gene in Saccharomyces cerevisiae. VMA1 consists of a single open reading frame and yet comprises two independent genetic information for Vma1p (a catalytic 70-kDa subunit of the vacuolar H+-ATPase) and VDE (a 50-kDa DNA endonuclease) as an in-frame spliced insert in the gene.

Morphological features of chick retinal ganglion cells.

On average, in chicks, the total number of retinal ganglion cells is 4.9 x 10(6) and the cell density is 10400 cells/mm2. Two high-density areas, namely the central area (CA) and the dorsal area (DA), are located in the central and dorsal retinas, respectively, in post-hatching day 8 (P8) chicks (19000 cells/mm2 in the CA; 12800 cells/mm2 in the DA).

Respiratory response of cell-based sensors to toxicants measured by using pseudo-random signals.

Cell-based sensors using such as Pseudomonas putida and Bacillus subtilis were applied to examine the toxicity of chemicals. Both toxicant and substrate solutions were introduced into the sensor system according to M-series and anti-symmetric M-series pseudo-random binary signals, Xn (n=15, minimal pulse width 4 min, period 60 min) and Yn (n=30, minimal pulse width 4 min, period 120 min). Toxicants such as Ag+, formaldehyde, azide, and hypo-chlorite were used to demonstrate the proposed method.

Tetrahydrobiopterin uptake in supplemental administration: elevation of tissue tetrahydrobiopterin in mice following uptake of the exogenously oxidized product 7,8-dihydrobiopterin and subsequent reduction by an anti-folate-sensitive process.

In order to increase the tissue level of tetrahydrobiopterin (BH4), supplementation with 6R-tetrahydrobiopterin (6RBH4) has been widely employed. In this work, the effectiveness of 6RBH4 was compared with 7,8-dihydrobiopterin (7,8BH2) and sepiapterin by administration to mice. Administration of 6RBH4 was the least effective in elevating tissue BH4 levels in mice while sepiapterin was the best.

apg15-1, a UGA mutant allele in the Saccharomyces cerevisiae APG16 gene, and its suppression by a cytoplasmic factor.

Autophagy is a complex cellular process by which starving cells utilize cytoplasmic macromolecules as nutritional resources. In Saccharomyces cerevisiae, more than 15 genes are involved in this process and most of them have been cloned and characterized by now. But there remains a complementation group represented by a single mutation, apg15-1, unclear as to its molecular nature.

In vitro chemosensitivity assays of retinoblastoma cells.

The use of chemotherapy in treating retinoblastoma is presently increasing in importance. Because of the rarity of this disease, excellent in vitro chemosensitivity assays are necessary, not only for individualized testing but also for the development of chemotherapy regimens. It is instructive for improving such assays to discuss in vitro chemosensitivitly tests of tumor cells from clinical specimens according to a cell biological perspective.

Inhibition of vertebrate telomerases by the triphosphate derivatives of some biologically active nucleosides.

In order to clarify the effect of the base moiety of nucleotide analogs on telomerase inhibition, triphosphate derivatives of biologically active nucleosides, 3'-azido-3'-deoxythymidine (AZT), 2'-deoxy-2'-fluoroarafuranosylthymine (FaraT), acycloguanosine (ACG) and their guanine or thymine counterparts (AZdG, FaraG and ACT, respectively) were investigated. In all of the present cases, guanine derivatives showed more potent inhibition than their thymine counterparts.

Inhibition of vertebrate telomerases by the triphosphate derivatives of carbocyclic oxetanocin analogs.

Telomerase is a cellular endogenous reverse transcriptase that uses its internal RNA as a template for extension of the telomere repeat, thus maintaining telomere length. In order to clarify the susceptibility of telomerase to triphosphate derivatives of carbocyclic oxetanocins, inhibition by 9-[trans-trans-2,3-bis(hydroxymethyl)cyclobutyl]guanine triphosphate (C.OXT-GTP) and its methylene analog, 9-(cis-3-hydroxymethyl-2-methylenecyclobutyl)guanine triphosphate (m-C.

Synthesis of antisense oligonucleotides containing photocleavable protecting groups on the thymine bases and their photoinduced duplex formation.

Oligonucleotides containing photocleavable protecting groups at thymine bases were synthesized to induce the duplex formation by photo-irradiation. 6-Nitroveratryloxycarbonyl (NVOC) group was used for the photocleavable protecting group at N3 position of thymidine. An oligonucleotide containing NVOC groups (NVOC-ODN2:5'-dATG CAC CAT(NVOC) TCT(NVOC)GTC TGT-3') was synthesized by phosphoramidite method.

A simplified measurement of hydrated crystal densities of low melting points (low transition points) by solidifications of aqueous solutions.

A volume change method for measuring crystal densities is described. It allows the densities of unstable hydrated crystals at room temperature to be determined, by measurements of volume changes during the solidification of aqueous solutions. NaCl x 2H2O, KCl, MgSO4 x 12H2O and K2HPO4 x 6H2O were measured by the method and their densities (SE) are 1.

Inhibition of human telomerase by nucleotide analogues bearing a hydrophobic group.

Telomerase, which synthesizes telomeric DNA in eukaryotic cells, is classified as a reverse transcriptase. To clarify the susceptibility of telomerase to nucleoside 5'-triphosphates bearing a hydrophobic group on the base moiety, we studied the inhibitory effects of 2',3'-dideoxy-5-styryluridine 5'-triphosphate analogues and 9-(beta-D-arabinofuranosyl)-2-(p-n-butylanilino)purine 5'-triphosphate analogues on telomerase activity using a quantitative 'stretch PCR' assay. 2',3'-Dideoxy-5-styryluridine 5'-triphosphate (StddUTP) showed more potent inhibition than 2',3'-dideoxythymidine 5'-triphosphate (ddTTP).

Telomerase-inhibitory effects of sugar-modified nucleotide analogs.

Telomerase is an endogenous reverse transcriptase that uses its internal RNA moiety as a template for the synthesis of telomere repeats, thus maintaining telomere length. To study the susceptibility of telomerase to sugar-modified nucleotide analogs, inhibition by arabinofuranosylguanine 5'-triphosphate (araGTP), 3'-azido-2',3'-dideoxyguanosine 5'-triphosphate (AZdGTP), 2',3'-dideoxy-2'-fluoroarabino-furanosylguanine 5'-triphosphate (FaraGTP), and their thymine counterparts was investigated. Among these compounds, all dGTP analogs showed potent inhibitory activity against human telomerase.

Telomerase-inhibitory effects of the triphosphate derivatives of some biologically active nucleosides.

Telomerase is a ribonucleoprotein reverse transcriptase that uses its internal RNA moiety as a template for synthesis of telomere repeats. To clarify the susceptibility of telomerase to HIV-1 reverse transcriptase inhibitors (RT), we investigated the inhibitory effects of 3'-azido-3'-deoxythymidine 5'-triphosphate (AZTTP), which is known to be a potent HIV-1 RT inhibitor, and acyclovir triphosphate (ACVTP). Lineweaver-Burk plot analyses showed that the inhibition mode of these compounds was competitive with the substrate dNTP counterpart.

Occurrence, horizontal transfer and degeneration of VDE intein family in Saccharomycete yeasts.

VDE is a homing endonuclease gene originally discovered as an intervening element in VMA1s of Saccharomyces cerevisiae. There have been two independent subfamilies of VDE, one from S. cerevisiae strain X2180-1A and the other from Saccharomyces sp.

Proteasome-driven turnover of tryptophan hydroxylase is triggered by phosphorylation in RBL2H3 cells, a serotonin producing mast cell line.

We previously demonstrated in mast cell lines RBL2H3 and FMA3 that tryptophan hydroxylase (TPH) undergoes very fast turnover driven by 26S-proteasomes [Kojima, M., Oguro, K., Sawabe, K.

The effect of social interactions on circadian self-feeding rhythms in rainbow trout Oncorhynchus mykiss Walbaum.

Circadian feeding rhythms have been revealed in several fish species, but whether or not social interactions influence the expression of the rhythms remains largely unexplored. This paper reported such an exploration in rainbow trout. The experiment was conducted in two consecutive stages in two adjacent insulated rooms.

Freeze-thawing behaviour of highly concentrated aqueous alkali chloride-glucose systems.

The freeze-thawing behaviour of highly concentrated aqueous alkali chloride-glucose systems was investigated by differential scanning calorimetry (DSC). In the aqueous NaCl-glucose solution system, single or double glass transitions followed by the corresponding devitrification exotherms were observed during rewarming. In the aqueous KCl-glucose solution system, on the other hand, a single glass transition followed by an exotherm was observed during rewarming.

Inhibition of human telomerase by L-enantiomers of natural 2'-deoxyribonucleoside 5'-triphosphates.

In order to clarify whether L-enantiomers of natural 2'-deoxyribonucleoside 5'-triphosphates (dNTPs) are recognized by human telomerase, a quantitative telomerase assay based on the 'stretch PCR' method was developed and used for kinetic analysis. Among the four L-dNTPs, L-dTTP and L-dGTP inhibited telomerase activity and the others showed slight or no inhibitory effect. Lineweaver-Burk plot analysis showed that the inhibition mode L-dGTP was competitive with dGTP.

Localization of cytosolic NADP-dependent isocitrate dehydrogenase in the peroxisomes of rat liver cells: biochemical and immunocytochemical studies.

Two types of NADP-dependent isocitrate dehydrogenases (ICDs) have been reported: mitochondrial (ICD1) and cytosolic (ICD2). The C-terminal amino acid sequence of ICD2 has a tripeptide peroxisome targeting signal 1 sequence (PTS1). After differential centrifugation of the postnuclear fraction of rat liver homogenate, approximately 75% of ICD activity was found in the cytosolic fraction.