Amidox, an inhibitor of ribonucleotide reductase, potentiates the action of Ara-C in HL-60 human promyelocytic leukemia cells

Amidox (3,4-dihydroxybenzamidoxime), a new polyhydroxy-substituted benzoic acid derivative, is a potent inhibitor of the enzyme ribonucleotide reductase (RR), which catalyses the de novo synthesis of DNA. RR is considered to be an excellent target for cancer chemotherapy. In the present study we investigated the antineoplastic effects of Amidox alone and in combination with Arabinofuranosylcytosine (Ara-C) in HL-60 human promyelocytic leukemia cells. In growth inhibition experiments Amidox yielded an IC50 of 30 microM, colony formation was inhibited at an IC50 of 20 microM as determined by a soft agar assay. Exposure of the cells to 75 and 100 microM Amidox for 24 hours was shown to significantly decrease intracellular dCTP, dGTP and dATP pools, whereas dTTP concentration increased, as determined by HPLC. The combination of Amidox with Ara-C yielded more than additive cytotoxic effects both in growth inhibition assays and in soft agar assays. We could show that--after preincubating the cells with 75 and 100 microM Amidox and subsequent exposure to Ara-C--intracellular Ara-CTP levels increased by 576% and 1143%, respectively. In conclusion, Amidox might offer an additional option for the treatment of leukemia and thus be further investigated in vitro and in vivo.     

Combination chemotherapy of BCNU and Didox acts synergystically in 9L glioma cells

Compounds inhibiting DNA repair and synthesis are expected to act synergistically with BCNU, a standard agent in the therapy of glioblastoma multiforme, and improve survival of patients with malignant gliomas. Ribonucleotide reductase (EC1.17.4.1; RR) catalyzes the rate-limiting step in DNA synthesis and plays a critical role in maintaining crucial substrates for DNA repair. We have studied the effects of Didox, an inhibitor of RR on 9L glioma cells in combination with BCNU. We analyzed intracellular dNTP pools and found that Didox significantly depleted the intracellular dNTP concentrations. Experiments using cytotoxicity, growth inhibition and clonogenic assays showed significant synergism of Didox and BCNU. Combination regimens using synchronous administration demonstrated highest cytotoxicity. We have also identified altered gene expression in a number of DNA repair related enzymes after BCNU treatment using large-scale cDNA arrays. The coadministration with Didox could reverse the expression of some of the overexpressed repair gene suggesting possible pathways to circumvent the developing resistance in 9L glioma cells against BCNU. These results introduce the combination of Didox and BCNU as a viable alternative for the treatment of malignant gliomas.     

Localisation of the Vacuolar Proton Pump (V-H+-ATPase) and Carbonic Anhydrase II in the Human Eccrine Sweat Gland

The localisation of the vacuolar proton pump (V-H+ -ATPase) and the enzyme carbonic anhydrase II (CAII) was investigated in the human eccrine sweat gland employing standard immunohistochemical techniques after antigen retrieval using microwave heat treatment and high pressure. The high-pressure antigen retrieval unmasked the presence of V-H+ -ATPase in the clear cells of the secretory coil, with a distribution similar to that previously observed for CAII. However, the dark cells were unreactive to both antibodies. In addition, heat and high-pressure antigen retrieval demonstrated the presence of CAII in the apical zone of luminal cells of the reabsorptive duct, a location not previously reported. The localisation of V-H+ -ATPase and CAII in the secretory coil clear cells suggests that the formation of HCO3- and H+ by carbonic anhydrase II and the transport of H+ by V-H+ -ATPase may play an role in sweat fluid secretion. Their presence at the apex of the duct cells indicates involvement in ductal ion reabsorption.     

Instructions for authors

Authors Index

AUTHOR INDEX VOLUME 170 2004     

Identification of human L-fucose kinase amino acid sequence

Fucose is a major component of complex carbohydrates. L-Fucose kinase (fucokinase) takes part in the salvage pathway for reutilization of fucose from the degradation of oligosaccharides. The amino acid sequence of human fucokinase was derived from a cDNA encoding a protein of hitherto unidentified function. Human fucokinase polypeptide chain consists of 990 amino acids with a predicted molecular mass of 107 kDa. The C-terminal part of its amino acid sequence showed sequence motifs typical for sugar kinases. Fucokinase full-length protein and a deletion mutant lacking the first 363 amino acids of the N-terminus were expressed in Escherichia coli BL21 cells. Both proteins displayed fucokinase activity. These results reveal that the discovered cDNA encodes the fucokinase protein and they confirm that a functional kinase domain is located in the C-terminal part of the enzyme.     

The Exobasidiales: An evolutionary hypothesis

To gain insight in the phylogenetic relationships within the Exobasidiales, septal pore apparatus, host-parasite interactions, sori, hymenia, basidia, and nucleotide sequences from the 5′ terminal domain of the nuclear large subunit rRNA gene were studied and compared. The results of our molecular phylogenetic analyses correlate well with the morphological data and both reflect the distribution of parasites on several host groups. Thus, the Exobasidiales seem to be divided into four groups, which are distinguishable by basidial morphology and host range as follows: (i) the Exobasidiaceae parasitizing mainly Ericanae are characterized by an abaxial orientation of the hilar appendices of the ballistosporic basidiospores on the elongate basidia, (ii) the Cryptobasidiaceae occurring mainly on Lauraceae sporulate inside the host tissue with elongate gastroid basidia, (iii) the Brachybasidiaceae living on monocots are characterized by elongate basidia bearing two ballistosporic basidiospores with adaxially oriented hilar appendices, and (iv) the Graphiolaceae occurring on palms produce chains of gastroid basidia in distinct basidiocarps. The arrangement of the four groups and the tree topology within Exobasidium derived from the molecular analyses essentially parallel phylogenetic host relationships, suggesting cospeciation. Based on our results, however, the radiation of Exobasidium on Vaccinioideae cannot be explained by cospeciation alone. 1 Part 204 of the series “Studies in Heterobasidiomycetes” from the Botanical Institute, University of Tübingen     

Extremely low frequency electromagnetic fields and heat shock can increase microvesicle motility in astrocytes

The effect of extremely low frequency electromagnetic fields (EMF) on microvesicles was examined in rat astrocytes by video-enhanced microscopy in combination with a perfusable cell chamber. The EMF effect was compared with the effect of heat shock (HS) and with a combination of them both. The velocity of microvesicles was measured using image processing software (NIH Scion image 1.61). After exposure of astrocytes to EMF (50 Hz, 100microT, 1 h), the velocity of microvesicles in astrocytes increased from 0.32 +/- 0.03 microm/s (n = 120, 95% CI) in the untreated control group to 0.41 +/- 0.03 microm/s (n = 175, 95% CI). Fifteen minutes after HS (45 degrees C, 10 min) the microvesicles showed a velocity of 0.56 +/- 0.03 microm/s (n = 125, 95% CI). Combination of HS and EMF led to an increase in velocity up to 0.54 +/- 0.03 microm/s (n = 110, 95% CI). No significant difference between HS and HS+EMF was found. Compared to the untreated control group, the increased microvesicle velocity of the exposed cells might be a stress response of the cell. It is possibly a sign of intensified intracellular traffic required to adjust the metabolic needs.     

Expression of a chimeric retroviral-lipase mRNA confers enhanced lipolysis in a hibernating mammal

Hibernating mammals can survive several months without feeding by limiting their carbohydrate catabolism and using triacylglycerols stored in white adipose tissue (WAT) as their primary source of fuel. Here we show that a lipolytic enzyme normally found in the gut, pancreatic triacylglycerol lipase (PTL), is expressed in WAT of hibernating 13-lined ground squirrels (Spermophilus tridecemlineatus). PTL expressed in WAT is encoded by an unusual chimeric retroviral-PTL mRNA approximately 500 bases longer than the predominant PTL message found in other ground squirrel tissues. Seasonal measurements detect the chimeric mRNA and PTL enzymatic activity in WAT before and during hibernation, with both showing their lowest observed levels 1 wk after hibernation concludes in mid-March. PTL is expressed in addition to hormone-sensitive lipase, the enzyme typically responsible for hydrolysis of triacylglycerols in WAT. Because of the distinct catalytic and regulatory properties of both enzymes, this dual-triacylglycerol lipase system provides a means by which the fuel requirements of hibernating 13-lined ground squirrels can be met without interruption.     

He-Ne laser irradiation protects B-lymphoblasts from UVA-induced DNA damage

The effect of He-Ne laser (632.8 nm) pre-irradiation on UVA (343 nm)-induced DNA damage in the human B-lymphoblast cell line NC37 was investigated using the comet assay. He-Ne laser pre-irradiation was observed to result in a dose-dependent decrease in UVA-induced DNA damage. This effect was also found to be dependent on the incubation period between He-Ne laser pre-irradiation and the UVA exposure. Whereas the control cells with a higher DNA damage point to an initial ability of faster repair, both the control and the He-Ne laser pre-irradiated cells subsequently show the same rate of DNA repair. The results suggest that He-Ne laser irradiation protect the cells from UVA-induced DNA damage primarily through an influence on processes that prevent an initial DNA damage. Received: 10 April 2000 / Accepted: 24 November 2000