The eukaryotic UDP-N-acetylglucosamine pyrophosphorylases. Gene cloning, protein expression, and catalytic mechanism.

A search of the yeast data base for a protein homologous to Escherichia coli UDP-N-acetylglucosamine pyrophosphorylase yielded UAP1 (UDP-N-acetylglucosamine pyrophosphorylase), the Saccharomyces cerevisiae gene for UDP-N-acetylglucosamine pyrophosphorylase. The Candida albicans and human homologs were also cloned by screening a C. albicans genomic library and a human testis cDNA library, respectively.

Severe growth defect in mouse cells lacking the telomerase RNA component.

The ribonucleoprotein enzyme telomerase synthesizes telomeric DNA onto chromosome ends. Telomere length is maintained, by the presence of telomerase activity, in the vast majority of primary tumours and stem cells, suggesting that telomere maintenance is essential for cellular immortalization. Recently, the telomerase RNA component in human and mouse (TERC and Terc, respectively), a telomerase-associated protein TEP1/TLP1 (refs 6,7) and the human catalytic subunit protein TERT (refs 8,9) have been identified.

Screening for glycosylphosphatidylinositol (GPI)-dependent cell wall proteins in Saccharomyces cerevisiae.

Open reading frames in the genome of Saccharomyces cerevisiae were screened for potential glycosylphosphatidylinositol (GPI)-attached proteins. The identification of putative GPI-attached proteins was based on three criteria: the presence of a GPI-attachment signal sequence, a signal sequence for secretion and a serine- or threonine-rich sequence. In all, 53 ORFs met these three criteria and 38 were further analyzed as follows.

Tumor selective delivery of 5-fluorouracil by capecitabine, a new oral fluoropyrimidine carbamate, in human cancer xenografts.

Capecitabine (N4-pentyloxycarbonyl-5'-deoxy-5-fluorocytidine) is a novel fluoropyrimidine carbamate that is converted to 5-fluorouracil (5-FUra) by three enzymes located in the liver and tumors; the final step is the conversion of 5'-deoxy-5-fluorouridine (5'-dFUrd) to 5-FUra by thymidine phosphorylase in tumors. The present study compared the efficacy of capecitabine and 5-FUra at their maximum tolerated doses in CXF280, HCT116, COLO205, and WiDr human colon cancer xenograft models, and measured subsequent 5-FUra and 5'-dFUrd levels in tumors and in the plasma and muscle. Capecitabine was effective in the first three models, whereas 5-FUra was effective only in CXF280, which is a cell line highly susceptible to fluoropyrimidines.

[Rbf1 (RPG-box binding factor), a transcription factor involved in yeast-hyphal transition of Candida albicans].

The major fungal pathogen for fungal diseases which have become a major medical problem in the last few years is Candida albicans, which can grow both in yeast and hyphae forms. This ability of C. albicans is thought to contribute to its colonization and dissemination within host tissues.

Induction of thymidine phosphorylase activity and enhancement of capecitabine efficacy by taxol/taxotere in human cancer xenografts.

Thymidine phosphorylase (dThdPase) is an essential enzyme for the activation of the cytostatics capecitabine (N(4)-pentyloxycarbonyl-5'-deoxy-5-fluorocytidine) and its intermediate metabolite [5'-deoxy-5-fluorouridine (5'-dFUrd)] to 5-fluorouracil in tumors. We have tried to identify the best partners of capecitabine in combination therapy, such as dThdPase up-regulators, which may enhance the efficacy of this compound. Among various cytostatics studied with the WiDr human colon cancer xenograft model, Taxol, Taxotere, and mitomycin C greatly increased levels of human dThdPase in tumors, and cyclophosphamide slightly increased the enzyme level.

High susceptibility of human cancer xenografts with higher levels of cytidine deaminase to a 2'-deoxycytidine antimetabolite, 2'-deoxy-2'-methylidenecytidine.

2'-Deoxy-2'-methylidenecytidine (DMDC) is a new 2'-deoxycytidine (dCyd) antimetabolite. The present study compared its antitumor activities with those of 2',2'-difluorodeoxy-cytidine (gemcitabine) in 15 human cancer xenograft models. DMDC was highly resistant to cytidine (Cyd) deaminase, which deaminates the dCyd analogues to inactive molecules, whereas gemcitabine was susceptible to the enzyme.

Apomorphine-induced hypoattention in rats and reversal of the choice performance impairment by aniracetam.

Aging-, disease- and medication-related imbalance of central dopaminergic neurons causes functional impairment of cognition and neuropsychological delirium in humans. We attempted to develop a new delirium model using the direct dopamine agonist, apomorphine, and a choice reaction performance task performed by middle-aged rats. The psychological properties of the model were assessed by determining behavioral measures such as choice reaction time, % correct and % omission.

The antiproliferative activity of DMDC is modulated by inhibition of cytidine deaminase.

We showed that the efficacy of the new 2'-deoxycytidine (2'-dCyd) analogue antimetabolite 2'-deoxy-2'-methylidenecytidine (DMDC) correlates well with tumor levels of cytidine (Cyd) deaminase in human cancer xenograft models. DMDC was highly effective in tumors with higher levels of Cyd deaminase, whereas lower levels yielded only slight activity. In contrast, gemcitabine (2',2'-difluorodeoxycytidine), which has action mechanisms similar to those of DMDC, is only slightly active in tumors with higher levels of the enzyme.

Flow cytometric analysis for sperm viability and counts in rats treated with trimethylphosphate or pyridoxine.

Six-week old SD-Slc male rats were treated for 4 weeks with compounds known to induce toxicological changes in male reproductive organs (pyridoxine in saline, 500 mg/kg/day, i.p.) or sperm (trimethylphosphate in distilled water, 100 mg/kg/day, p.

Isolation of CaSLN1 and CaNIK1, the genes for osmosensing histidine kinase homologues, from the pathogenic fungus Candida albicans.

Recent studies have revealed that fungi possess a mechanism similar to bacterial two-component systems to respond to extracellular changes in osmolarity. In Saccharomyces cerevisiae, Sln1p contains both histidine kinase and receiver (response regulator) domains and acts as an osmosensor protein that regulates the downstream HOG1 MAP kinase cascade. SLN1 of Candida albicans was functionally cloned using an S.

Positive correlation between the efficacy of capecitabine and doxifluridine and the ratio of thymidine phosphorylase to dihydropyrimidine dehydrogenase activities in tumors in human cancer xenografts.

Capecitabine (N4-pentyloxycarbonyl-5'-deoxy-5-fluorocytidine) is a new fluoropyrimidine carbamate, which is converted to 5-fluorouracil (5-FUra) selectively in tumors through the intermediate metabolite 5'-deoxy-5-fluorouridine (5'-dFUrd, doxifluridine). 5'-dFUrd is metabolized to 5-FUra by thymidine phosphorylase (dThdPase) located in high levels in various types of solid tumors from patients, whereas 5-FUra generated is catabolized to dihydrofluorouracil by dihydropyrimidine dehydrogenase (DPD). The present study investigated whether the efficacy of capecitabine and its intermediate metabolite 5'-dFUrd correlates with levels of these enzymes in various human cancer xenograft models.

Cloning of the RHO1 gene from Candida albicans and its regulation of beta-1,3-glucan synthesis.

The Saccharomyces cerevisiae RHO1 gene encodes a low-molecular-weight GTPase. One of its recently identified functions is the regulation of beta-1,3-glucan synthase, which synthesizes the main component of the fungal cell wall (J. Drgonova et al.

A solid-phase assay to screen monoclonal antibodies against DNA-binding protein.

A method is described for selecting monoclonal antibodies (mAb) against DNA-binding protein. The protocol involves a non-radioactive solid-phase DNA binding assay using a 96-well plate. Because the solid-phase assay is highly specific and sensitive, partially purified antigen is sufficient for the immunization, and mAb screening can be performed with crude cell extract as the antigen.

[Efficacy of combination chemotherapy of cyclophosphamide and 5'-deoxy-5-fluorouridine in a mammary tumor xenograft model, MX-1].

Efficacy of long-term combination chemotherapy of cyclophosphamide (CPA) and 5'-deoxy-5-fluorouridine (5'-DFUR), both of which have been widely used as chemotherapeutics against breast cancer patients, was examined in a mammary tumor xenograft model, MX-1. 5'-DFUR suppressed the tumor growth over a long period and prolonged the survival, although it did not reduce the initial tumor burden, CPA induced the disappearance of the tumor burden temporarily. However, CPA became inaffective despite continuation of treatment, and induced the recurrence of the tumor.

Regulation by tetracycline of gene expression in Saccharomyces cerevisiae.

A convenient system for the control of gene expression in Saccharomyces cerevisiae was developed. Tetracycline-responsive promoters were constructed by fusing the tetracycline operator (tetO) to the S. cerevisiae HOP1 promoter.

Cloning of the Candida albicans homolog of Saccharomyces cerevisiae GSC1/FKS1 and its involvement in beta-1,3-glucan synthesis.

Saccharomyces cerevisiae GSC1 (also called FKS1) and GSC2 (also called FKS2) have been identified as the genes for putative catalytic subunits of beta-1,3-glucan synthase. We have cloned three Candida albicans genes, GSC1, GSL1, and GSL2, that have significant sequence homologies with S. cerevisiae GSC1/FKS1, GSC2/FKS2, and the recently identified FKSA of Aspergillus nidulans at both nucleotide and amino acid levels.

[Immune intervention by peptides having a MHC class II binding motif--application of MHC blockers to autoimmune disease models].

Intervention of T cell activation and the treatment of autoimmune disease models by MHC class II binding peptides was reviewed in this article. Analog peptides derived from antigenic peptides were shown to inhibit T cell activation in vitro as well as in vivo either by T cell antagonism, T cell tolerance induction, or by MHC blockade. The induction of immune suppression by MHC blocker peptides was discussed in detail.

Cyclothialidine analogs, novel DNA gyrase inhibitors.

DNA gyrase inhibitors, cyclothialidines B, C, D and E were isolated from four Streptomycete strains (NR 0659, NR 0660, NR 0661 and NR 0662). Their structures have been elucidated based on the amino acid analysis of the hydrolysates, NMR and HRFAB-MS experiments and shown to be cyclothialidine analogs. The absolute stereochemistry has been determined by the chiral HPLC analysis of the hydrolysates.

Properties of yeast expressed Aspergillus nidulans chitin synthase B which is essential for hyphal growth.

A complementary DNA of the Aspergillus nidulans chsB gene encoding chitin synthase, an essential gene for hyphal growth, was obtained by RT-PCR and expressed in Saccharomyces cerevisiae by using the GAL1 promoter in a multicopy plasmid. The biochemical characteristics of chitin synthase B (ChsB) expressed in S. cerevisiae were examined.

The phylogeny of the genera Chryseomonas, Flavimonas, and Pseudomonas supports synonymy of these three genera.

The 16S rRNA sequences of Chryseomonas luteola, the type species of the genus Chryseomonas, and Flavimonas oryzihabitans, the type species of the genus Flavimonas, were determined. These sequences were compared with the sequences of 27 representative strains of the genus Pseudomonas. C.

Isolation of the Candida albicans homologs of Saccharomyces cerevisiae KRE6 and SKN1: expression and physiological function.

Cell wall beta-glucan in a pathogenic fungus, Candida albicans, is highly branched with beta-1,3 and beta-1,6 linkages. We have isolated the C. albicans cDNAs for KRE6 and SKN1, the genes required for beta-1,6-glucan synthesis in Saccharomyces cerevisiae.

Inactivation of the cyclin D-dependent kinase in the rat fibroblast cell line, 3Y1, induced by contact inhibition.

Cyclin-dependent kinase (Cdk) inhibitory proteins are involved in cell cycle arrest induced by antiproliferating factors or chemicals. High cell density also induces cell cycle arrest in which the genomic DNA is unreplicated, even in the presence of a mitotic dose of growth factors; this is termed contact inhibition. Although the cell cycle of the rat fibroblast cell line, 3Y1, was arrested in quiescence by contact inhibition, the Cdk4 bound to its regulatory subunit, cyclin D1 or D3.

Biochemical and genetic characterization of Rbf1p, a putative transcription factor of Candida albicans.

A Candida albicans gene encoding a novel DNA-binding protein that bound to the RPG box of Saccharomyces cerevisiae and the telomeric repeat sequence of C. albicans was previously cloned and designated RBF1 (RPG-box-binding factor). In this report, determination of the functional domains of the protein is described.