[Discovery and development of novel anticancer drug capecitabine].

Capecitabine (N4-pentyloxycarbonyl-5'-deoxy-5-fluorocytidine) is a novel oral fluoropyrimidine carbamate, which was designed to be sequentially converted to 5-fluorouracil (5-FU) by three enzymes located in the liver and in tumors. N4-alkoxycarbonyl-5'-deoxy-5-fluorocytidine derivatives including capecitabine pass intact through the intestinal tract and are sequentially converted to 5-FU by a cascade of the three enzymes. The first step is the conversion to 5'-deoxy-5-fluorocytidine (5'-DFCR) by carboxylesterase located in the liver, then to 5'-deoxy-5-fluorouridine (5'-DFUR) by cytidine deaminase highly expressed in the liver and various solid tumors, and finally to 5-FU by thymidine phosphorylase (dThdPase) preferentially located in tumor tissues.

Cdk4 activation is dependent on the subunit rearrangement in the complexes.

Although several factors have been implicated in the regulation of Cdk4 activity, little is known regarding the contributions of cyclin-dependent kinase inhibitors (CKIs) in Cdk4 activation in the mid G1 phase. Using a mouse macrophage cell line (Bac1.2F5), we found that most of Cdk4 bound to p15 when cells were in a quiescent state.

Prenylation of Rho1p is required for activation of yeast 1, 3-beta-glucan synthase.

One of the essential protein substrates of geranylgeranyl transferase type I in the budding yeast Saccharomyces cerevisiae is a rho-type GTPase, Rho1p, which is a regulatory subunit of 1, 3-beta-glucan synthase. Previous studies have indicated that modification of Rho1p is significantly reduced in a mutant of the beta subunit of geranylgeranyl transferase type I called cal1-1. Here we present genetic and biochemical evidence showing that modification of Rho1p is required for activity of 1,3-beta-glucan synthase.

Application of homogeneous time-resolved fluorescence (HTRFTM) to monitor poly-ubiquitination of wild-type p53.

Rapid degradation of wild-type p53 in the human uterine cervix is induced by the infection of high-risk human papilloma virus (HPV) types 16 and 18. HPV-E6 protein plays a critical role in the poly-ubiquitination of wild-type p53 by mediating the association of p53 with E6-associated protein (E6AP). As a result, the poly-ubiquitinated p53 is rapidly and selectively degraded by the 26S proteasome.

The Candida albicans gene for mRNA 5-cap methyltransferase: identification of additional residues essential for catalysis.

The 5'-cap structure of eukaryotic mRNA is methylated at the terminal guanosine by RNA (guanine-N7-)-methyltransferase (cap MTase). Saccharomyces cerevisiae ABD1 (ScABD1) and human hMet (also called CMT1) genes are responsible for this enzyme. The ABD1 homologue was cloned from the pathogenic fungus Candida albicans and named C.

X-ray irradiation induces thymidine phosphorylase and enhances the efficacy of capecitabine (Xeloda) in human cancer xenografts.

Thymidine phosphorylase (dThdPase) is an essential enzyme for the activation of the cytostatics capecitabine (N4-pentyloxycarbonyl-5'-deoxy-5-fluorocytidine) and its intermediate metabolite 5'-deoxy-5-fluorouridine (5'-dFUrd) to 5-fluorouracil (5-FUra) in tumors. We observed previously that several cytokines and cytostatics up-regulated dThdPase expression and consequently enhanced the efficacy of capecitabine and 5'-dFUrd. In the present study, we found that X-ray irradiation also up-regulated dThdPase expression in several human cancer xenografts.

Activation of the reticulothalamic cholinergic pathway by the major metabolites of aniracetam.

The aim of the study was to further investigate the effects of aniracetam, a cognition enhancer, and its metabolites on the brain cholinergic system. We measured choline acetyltransferase activity and acetylcholine release using in vivo brain microdialysis in stroke-prone spontaneously hypertensive rats (SHRSP). The enzyme activity in the pons-midbrain and hippocampus, and basal acetylcholine release in the nucleus reticularis thalami were lower in SHRSP than in age-matched Wistar Kyoto rats, indicating central cholinergic deficits in SHRSP.

Induction of thymidine phosphorylase expression and enhancement of efficacy of capecitabine or 5'-deoxy-5-fluorouridine by cyclophosphamide in mammary tumor models.

Thymidine phosphorylase (dThdPase) is an essential enzyme for the activation of the oral cytostatic drugs capecitabine (N(4)-pentyloxycarbonyl-5'-deoxy-5-fluorocytidine, Xeloda(trade mark)) and its intermediate metabolite doxifluridine [5'-deoxy-5-fluorouridine (5'-dFUrd, Furtulon((R)))] to 5-fluorouracil (5-FUra) in tumors. In a previous study, we found that several cytostatics were able to up-regulate tumor levels of dThdPase in a human colon cancer xenograft model. In the present study, we confirmed that the administration of cytostatics used for breast cancer treatment, such as taxanes and cyclophosphamide (CPA), up-regulated the tumor level of dThdPase in mammary tumor models as well.

The Candida albicans CHS4 gene complements a Saccharomyces cerevisiae skt5/chs4 mutation and is involved in chitin biosynthesis.

The Candida albicans CHS4 gene encoding chitin synthase 4 has been isolated using the Saccharomyces cerevisiae CHS4/SKT5 gene as a probe. The gene contains a 2061 bp open reading frame capable of encoding a protein of 687 amino acids (76053 Da). No intron was observed in the gene.

Amino acid residues in the omega-minus region participate in cellular localization of yeast glycosylphosphatidylinositol-attached proteins.

The final destination of glycosylphosphatidylinositol (GPI)-attached proteins in Saccharomyces cerevisiae is the plasma membrane or the cell wall. Two kinds of signals have been proposed for their cellular localization: (i) the specific amino acid residues V, I, or L at the site 4 or 5 amino acids upstream of the GPI attachment site (the omega site) and Y or N at the site 2 amino acids upstream of the omega site for cell wall localization and (ii) dibasic residues in the region upstream of the omega site (the omega-minus region) for plasma membrane localization. The relationships between these amino acid residues and efficiencies of cell wall incorporation were examined by constructing fusion reporter proteins from open reading frames encoding putative GPI-attached proteins.

Epidemiology of visceral mycoses: analysis of data in annual of the pathological autopsy cases in Japan.

The data on visceral mycoses that had been reported in the Annual of the Pathological Autopsy Cases in Japan from 1969 to 1994 by the Japanese Society of Pathology were analyzed epidemiologically. The frequency of visceral mycoses among the annual total number of pathological autopsy cases increased noticeably from 1.60% in 1969 to a peak of 4.

Tumour inoculation site-dependent induction of cachexia in mice bearing colon 26 carcinoma.

Murine colon 26 carcinoma growing at either subcutaneous (s.c.) or intramuscular (i.

Advantages of toxicokinetics in new drug development.

Toxicokinetic (TK) study is generally required for toxicological evaluation and safety assessment, particularly in the pivotal toxicological studies which are requested on the basis of drug registration. The estimation points of TK data are: (1) determination of TK profile in toxicity study, (2) selection of dose, dosing form, alternative dosing route, (3) help for the evaluation of toxicity (non-effect/toxic dose, toxicological mechanism), (4) comparative evaluation between animal and human cases, (5) recommendation of the starting dose in the first human clinical trial. On the other hand, as a very recent trend, TK data are practically used for the purpose of drug discovery such as lead-optimization and candidate-selection.

Mutational analysis of chitin synthase 2 of Saccharomyces cerevisiae. Identification of additional amino acid residues involved in its catalytic activity.

Saccharomyces cerevisiae harbors three chitin synthases termed Chs1p, Chs2p and Chs3p. Previously, we demonstrated that con1, a region that is highly conserved among all chitin synthases, contains amino acids essential for the catalytic activity of the enzyme and that Asp562, Gln601, Arg604, and Trp605 found in con1 together with Asp441 were probable catalytic sites of the enzyme. Here we report that another region, con2, in the C-terminal half of Chs2p is also conserved exclusively in chitin synthases that resemble S.

Synthesis of novel antifungal agents (2).

Synthesis of novel cyclohexyl analogs of restricticin using intramolecular radical cyclization and their in vitro and in vivo antifungal activities are described.

Modeling, synthesis and biological activity of novel antifungal agents (1).

Homology modeling of candida lanosterol C-14 demethylase, synthesis and in vitro antifungal activities of cyclohexyl analogs of restricticin are described.

Isolation and Characterization of a New Quinoprotein Dehydrogenase, L-Sorbose/L-Sorbosone Dehydrogenase.

Gluconobacter oxydans DSM 4025 effectively oxidizes L-sorbose to 2-keto-L-gulonic acid (2 KGA), an industrial precursor of vitamin C. From this microorganism, we purified the enzyme involved in this oxidation reaction. The enzyme is a unique quinoprotein dehydrogenase catalyzing not only the conversion of L-sorbose to L-sorbosone, but also that of L-sorbosone to 2 KGA.

Production of Vitamin B6 in Rhizobium.

The production of vitamin B6 was studied in about 1,590 bacterial isolates from soil, and an isolate, 28-21, identified as Rhizobium leguminosarum was obtained as a vitamin B6 high producer. Then, the production of vitamin B6 by commercially available Rhizobium strains was examined, and many of the tested strains excreted large amounts of vitamin B6 into the culture broth. The best producer of vitamin B6 was R.

Saccharomyces cerevisiae GNA1, an essential gene encoding a novel acetyltransferase involved in UDP-N-acetylglucosamine synthesis.

The Saccharomyces cerevisiae gene, YFL017C, for a putative acetyltransferase was characterized. Disruption of YFL017C was lethal, leading to a morphology similar to those caused by the depletion of AGM1 or UAP1, the genes encoding phospho-N-acetylglucosamine mutase and UDP-N-acetylglucosamine pyrophosphorylase, respectively. This implies the involvement of YFL017C in UDP-N-acetylglucosamine synthesis.

A controllable gene-expression system for the pathogenic fungus Candida glabrata.

A system for controlling gene expression was established in the pathogenic fungus Candida glabrata to elucidate the physiological functions of genes. To control the expression of the gene of interest, the C. glabrata cells were first transformed with the plasmid carrying the tetracycline repressor-transactivator fusion tetR::GAL4, then with the DNA fragment containing the controllable cassette, the tetracycline operator chimeric promoter (tetO::ScHOP1).

Amino acid sequence requirement for efficient incorporation of glycosylphosphatidylinositol-associated proteins into the cell wall of Saccharomyces cerevisiae.

During cell wall biogenesis in Saccharomyces cerevisiae, some glycosylphosphatidylinositol (GPI)-attached proteins are detached from GPI moieties and bound to beta-1,6-glucan of the cell wall. The amino acid sequence requirement for the incorporation of GPI-attached proteins into the cell wall was studied by using reporter fusion proteins. Only the short omega-minus region composed of five amino acids, which is located upstream of the omega site for GPI attachment, determined the cellular localization of the GPI-associated proteins.

A second p53-related protein, p73L, with high homology to p73.

The p53 protein, which regulates the rate of cell division and death, is the most frequently mutated tumor suppressor to be identified so far in human cancers. Recently, a gene with significant homology to p53, termed p73, has been identified in a chromosomal region that is implicated in the molecular pathogenesis of neuroblastoma. We have cloned a second human p53-related gene, termed p73L, which shows strong amino-acid similarity to p73.

Antitumor activities of a novel fluoropyrimidine, N4-pentyloxycarbonyl-5'-deoxy-5-fluorocytidine (capecitabine).

Capecitabine (N4-pentyloxycarbonyl-5'-deoxy-5-fluorocytidine) is a novel fluoropyrimidine carbamate that was synthesized for the purpose of finding antitumor drugs with improved safety and efficacy profiles compared with those of 5-fluorouracil (5-FUra) and doxifluridine (5'-deoxy-5-fluorouridine, 5'-dFUrd). The present study compared the antitumor activities of the compound with those of other fluoropyrimidines in 12 human cancer xenograft models and their antimetastatic activities in murine tumor models. The antitumor efficacy of capecitabine was greater than those of 5'-dFUrd, UFT (a mixture of tegafur and uracil) and 5-FUra.

Screening of an inhibitor of the tetracycline efflux pump in a tetracycline-resistant clinical-isolate of Staphylococcus aureus 743.

Clinically-isolated methicillin-resistant Staphylococcus aureus (MRSA) strain 743 exhibited resistance to tetracycline as judged from the active efflux of the drug. The efflux of tetracycline was inhibited by an uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and minocycline. Inhibitors of the efflux pump were examined in this strain to determine the cellular accumulation of tetracycline.

Regulation of Vbeta germline transcription in RAG-deficient mice by the CD3epsilon-mediated signals: implication of Vbeta transcriptional regulation in TCR beta allelic exclusion.

During the thymic development of alphabeta lineage T cells, maturation of the CD4- CD8- double-negative (DN) cells into the CD4+ CD8+ double-positive cells is accompanied by the induction of TCR beta allelic exclusion. Recent studies have shown that these events are regulated by the signals through the pre-TCR complex which consists of the TCR beta, pre-TCR alpha and CD3 components. The Vbeta germline transcripts are detected prior to the TCR beta chain gene rearrangements in the DN thymocytes.