Inhibition of human telomerase by 7-deaza-2'-deoxyguanosine nucleoside triphosphate analogs: potent inhibition by 6-thio-7-deaza-2'-deoxyguanosine 5'-triphosphate

We have examined analogs of the previously reported 7-deaza-2'-deoxypurine nucleoside triphosphate series of human telomerase inhibitors. Two new telomerase-inhibiting nucleotides are reported: 6-methoxy-7-deaza-2'-deoxyguanosine 5'-triphosphate (OMDG-TP) and 6-thio-7-deaza-2'-deoxyguanosine 5'-triphosphate (TDG-TP). In particular, TDG-TP is a very potent inhibitor of human telomerase with an IC(50) of 60 nM. TDG-TP can substitute for dGTP as a substrate for telomerase, but only at relatively high concentrations. Under conditions in which TDG-TP is the only available guanosine substrate, telomerase becomes nonprocessive, synthesizing short products that appear to contain only one to three TDG residues. Similarly, the less potent telomerase inhibitor OMDG-TP gives rise to short telomerase products, but less efficiently than TDG-TP. We show here that TDG-TP, and to a lesser extent OMDG-TP, can serve as substrates for both templated (Klenow exo) and nontemplated (terminal transferase) DNA polymerases. For either polymerase, the products arising from TDG-TP are relatively short, and give rise to bands of unusual mobility under PAGE conditions. These anomalous bands revert, under treatment with DTT, to normal mobility bands, indicating that these products may contain thiol-labile disulfide linkages involving the incorporated TDG residues. This observation of potential TDG-crosslinks may have bearing on the mechanism of telomerase inhibition by this nucleotide analog.     

Effect of 6-Hydroxydopamine Lesions of the Medial Prefrontal Cortex on Neurotransmitter Systems in Subcortical Sites in the Rat

The effect of lesions of the catecholamine nerve terminals in the medial prefrontal cortex of the rat on neurotransmitter mechanisms within the basal ganglia has been investigated. Bilateral 6-hydroxydopamine lesions were stereotaxically placed in the dopamine-rich (DA) area of th frontal cortex. Animals were pretreated with desmethylimipramine to block the uptake of neurotoxin into noradrenergic (NA) terminals and to make it more selective for DA terminals. The lesion produced a selective reduction of both NA and DA from the medial prefrontal cortex, a result related to falls in tyrosine hydroxylase activity at this site. Lesioned animals showed enhanced DA turnover and utilisation in striatal and limbic regions. There was no change in subcortical tyrosine hydroxylase activity. In addition there were significant falls in other putative neurotransmitters within basal sites, including 5-hydroxytryptamine and GABA. Decreased activity of the neurotransmitter-synthesizing enzyme glutamate decarboxylase and choline acetyltransferase was also recorded in certain regions of the basal ganglia. The results suggest that frontal cortical catecholamine systems may serve to regulate various neurotransmitter mechanisms in the basal ganglia.     

Synthesis and evaluation of anticancer benzoxazoles and benzimidazoles related to UK-1

UK-1 is a structurally unique bis(benzoxazole) natural product isolated from a strain of Streptomyces. UK-1 has been reported to possess anticancer activity but no activity against bacteria, yeast, or fungi. Previous work has also demonstrated the ability of UK-1 to bind a variety of di- and tri-valent metal ions, particularly Mg²⁺ ions, and to form complexes with double-stranded DNA in the presence of Mg²⁺ ions. Here we report the activity of UK-1 against a wide range of human cancer cell lines. UK-1 displays a wide spectrum of potent anticancer activity against leukemia, lymphoma, and certain solid tumor-derived cell lines, with IC₅₀ values as low as 20 nM, but is inactive against Staphylococcus aureus, a methicillin-resistant strain of S. aureus, or Pseudomonas aeruginosa. A series of analogues of the bis(benzoxazole) natural product UK-1 in which the carbomethoxy-substituted benzoxazole ring of the natural product was modified were prepared and evaluated for their anticancer and antibacterial properties. An analogue of UK-1 in which the carbomethoxy-substituted benzoxazole ring was replaced with a carbomethoxy-substituted benzimidazole ring was inactive against human cancer cell lines and the two strains of S. aureus. In contrast, a simplified analogue in which the carbomethoxy-substituted benzoxazole ring was replaced with a carbomethoxy group was almost as active as UK-1 against the four cancer cell lines examined but lacked activity against S. aureus. Metal ion binding studies of these analogues demonstrate that they both bind Zn²⁺ and Ca²⁺ ions about as well as UK-1. The non-cytotoxic benzimidazole UK-1 analogue binds Mg²⁺ ions 50-fold weaker than UK-1, whereas the simple benzoxazole analogue binds Mg²⁺ ions nearly as well as UK-1. These results support a role of Mg²⁺ ion binding in the selective cytotoxicity of UK-1 and provide a minimal pharmacophore for the selective cytotoxic activity of the natural product.     

A tunable hydrogel for encapsulation and controlled release of bioactive proteins

A N,N-dimethylacrylamide-based hydrogel (2) with the new cross-linker (ethylenedioxy) bis[2,2'-(N-acryloylamino)ethane] (1) has been prepared, and its physicochemical properties in aqueous solution were studied. Three different native proteins (lysozyme, bovine serum albumin, and rabbit IgG) were encapsulated within the polymeric matrix 2, and the kinetics of their release from the swollen hydrogel were determined. The rate of protein release exhibits a clear dependence on both the molecular weight of the protein and the amount of cross-linker utilized to prepare the hydrogel. This is reflected by the fact that the low molecular weight proteins are released at an increased rate versus higher molecular weight proteins. In addition a greater amount of protein is released from the hydrogels with a lower percentage of cross-linker. The polymerization procedure used in this study is sufficiently mild to safeguard the functional integrity of attendant biomolecules as determined by the retention of catalytic activity of encapsulated alpha-chymotrypsin and aldolase catalytic antibody 38C2. The potential utility of these hydrogels for the controlled release of bioactive agents in vivo is strengthened by both their lack of toxicity against human dermal fibroblasts and their lack of immunogenicity in mice.     

Carbohydrate-mediated Recognition of Larval Mosquito Hosts by Romanomermis culicivorax

Proteases, glycosidases, and lectins were tested and the results supported a role in host recognition for glycoproteins containing β-glucose and α-mannose on the cuticular surface of host and parasite. Carbohydrates containing α-glucose, galactose, fucose, or N-acetylglucosamine residues apparently are not involved in nematode attachment. Chitin or a related N-acetylglucosamine polymer was found in R. culicivorax preparasites. Treatment of preparasites with neuraminidase, which hydrolyzes sialic acids, increased nematode attachment to Anopheles freeborni larvae.     

Biological aspects of the interaction between Coelomomyces psorophorae zygotes and the larvae of Culiseta inornata: Environmental factors

Zygotes of the obligately parasitic watermold Coelomomyces psorophorae encyst on the cuticular surface of susceptible mosquito larvae as the initial step in the infection process. Zygotes can successfully encyst on Culiseta inornata larvae at temperatures from 12°–30°C, with an optimum of 25°C. Optimum encystment on larvae occurs from pH 6.5–8.5, with activity documented from pH 5.0–9.5. Zygotes are active in water with total salinity of 5.4 ppt down to double glass-distilled water.     

Duplex and quadruplex DNA binding and photocleavage by trioxatriangulenium ion

The stable trioxatriangulenium ion (TOTA) has previously been shown to bind to and photooxidize duplex DNA, leading to cleavage at G residues, particularly 5'-GG-3' repeats. Telomeric DNA consists of G-rich sequences that may exist in either duplex or G-quadruplex forms. We have employed electrospray ionization mass spectrometry (ESI-MS) to investigate the interactions between TOTA and duplex DNA or G-quadruplex DNA. A variety of duplex decamer oligodeoxynucleotides form complexes with TOTA that can be detected by ESI-MS, and the stoichiometry and fragmentation patterns observed are commensurate with an intercalative binding mode. TOTA also forms complexes with four-stranded and hairpin-dimer G-quadruplex oligodeoxynucleotides that can be detected by ESI-MS. Both the stoichiometry and the fragmentation patterns observed by ESI-MS are different than those observed for G-tetrad end-stacking binding ligands. We have carried out (1)H NMR titrations of a four-stranded G-quadruplex in the presence of TOTA. Addition of up to 1 equiv of TOTA is accompanied by pronounced upfield shifts of the G-tetrad imino proton resonances in the NMR, which is similar to the effect observed for G-tetrad end-stacking ligands. At higher ratios of added TOTA, there is evidence for additional binding modes. Duplex DNA containing either human telomeric repeats (T(2)AG(3))(4) or the Tetrahymena telomeric repeats (T(2)G(4))(4) are readily photooxidized by TOTA, the major sites of oxidation being the central guanine residues in each telomeric repeat. These telomeric repeats were incorporated into duplex/quadruplex chimeras in which the repeats adopt a G-quadruplex structure. Analysis by denaturing polyacrylamide gel electrophoresis reveals significantly less TOTA photocleavage of these quadruplex telomeric repeats when compared to the duplex repeats.     

JAtlasView: a Java atlas-viewer for browsing biomedical 3D images and atlases

Background  

Many three-dimensional (3D) images are routinely collected in biomedical research and a number of digital atlases with associated anatomical and other information have been published. A number of tools are available for viewing this data ranging from commercial visualization packages to freely available, typically system architecture dependent, solutions. Here we discuss an atlas viewer implemented to run on any workstation using the architecture neutral Java programming language.