Accumulation and toxicity of monophenyl arsenicals in rat endothelial cells

Clark 1 (diphenylarsine chloride) and Clark 2 ( diphenylarsine cyanide) were used as chemical weapon agents (CWA), and the soil contamination by these CWA and their degraded products, diphenyl and phenyl arsenicals, has been one of the most serious environmental issues. In a series of comparisons in toxicity between trivalent and pentavalent arsenicals we investigated differences in the accumulation and toxicity of phenylarsine oxide (PAO(3+)) and phenylarsonic acid (PAA(5+)) in rat heart microvascular endothelial cells. Both the cellular association and toxicity of PAO(3+) were much higher than those of PAA(5+), and LC50 values of PAO(3+) and PAA(5+) were calculated to be 0.295 muM and 1.93 mM, respectively. Buthionine sulfoximine, a glutathione depleter, enhanced the cytotoxicity of both PAO(3+) and PAA(5+). N-Acetyl-L-cysteine (NAC) reduced the cytotoxicity and induction of heme oxygenase-1 (HO-1) mRNA in PAO(3+)-exposed cells, while NAC affected neither the cytotoxicity nor the HO-1 mRNA level in PAA(5+)-exposed cells. The effect of NAC may be due to a strong affinity of PAO(3+) to thiol groups because both NAC and GSH inhibited the cellular accumulation of PAO(3+), but PAA(3+) increased tyrosine phosphorylation levels of cellular proteins. These results indicate that the inhibition of protein phosphatases as well as the high affinity to cellular components may confer PAO(3+) the high toxicity.

Urinary arsenic speciation and porphyrins in C57B1/6J mice chronically exposed to low doses of sodium arsenate

Arsenic has been classified as a human carcinogen based on epidemiological data however the mechanism of its carcinogenicity is still unclear. Urinary biomarkers for chronic arsenic exposure would be valuable as an early warning indicator for timely interventions. In this study, young female C57BI/6J mice were given drinking water containing 0, 100, 250 and 500 mug As-v/L as sodium arsenate ad libitum for 12 months. Urine was collected bimonthly for urinary arsenic methylation assay and porphyrin analysis. All detectable arsenic species showed strong linear correlation with administered dosage and the arsenic methylation patterns were similar in all three treatment groups. No significant changes of methylation patterns were observed over time for either the control or test groups. Urinary coproporphyrin III was significantly increased in the 8th month in 250 and 500 mug/L groups and remained significantly dose-related after 10 and 12 months. Coproporphyrin I also showed a significant dose-response relationship after 12 months. Our results confirm that urinary arsenic is a useful biomarker for internal dose. The alteration of porphyrin profile suggests that arsenic can affect the heme metabolism and this may occur prior to the onset of arsenic induced carcinogenesis. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

HPP1: A transmembrane protein-encoding gene commonly methylated in colorectal polyps and cancers

Adenomas are the precursors of most colorectal cancers. Hyperplastic polyps have been linked to the subset of colorectal cancers showing DNA microsatellite instability, but little is known of their underlying genetic etiology. Using a strategy that isolates differentially methylated sequences from hyperplastic polyps and normal mucosa, we identified a 370-bp sequence containing the 5' untranslated region and the first exon of a gene that we have called HPP1. Rapid amplification of cDNA ends was used to isolate HPP1 from normal mucose. Using reverse transcription-PCR, HPP1 was expressed in 28 of 30 (93%) normal colonic samples but in only seven of 30 (23%) colorectal cancers (P < 0.001). The 5' region of HPP1 included a CpG island containing 49 CpG sites, of which 96% were found to be methylated by bisulfite sequencing of DNA from colonic tumor samples. By COBRA analysis, methylation was detected in six of nine (66%) adenomas, 17 of 27 (63%) hyperplastic polyps, and 46 of 55 (84%) colorectal cancers. There was an inverse relationship between methylation level and mRNA expression in cancers (r = -0.67; P < 0.001), and 5-aza-2-deoxycytidine treatment restored HPP1 expression in two colorectal cancer cell lines. In situ hybridization of HPP1 indicated that expression occurs in epithelial and stromal elements in normal mucosa but is silenced in both cell types in early colonic neoplasia. HPP1 is predicted to encode a transmembrane protein containing follistatin and epidermal growth factor-like domains. Silencing of HPP1 by methylation may increase the probability of neoplastic transformation.

Chemistry, properties, and phylogenetic implications of the methylated carrageenans from red algae of the genus Areschougia (Areschougiaceae, Gigartinales, Rhodophyta)

The three Australian-endemic species comprising the genus Aresehougia have been examined to determine the structure of their nonfibrillar wall components. The polysaccharide extracted from the most widely distributed species, A. congesta (Turner) J. Agardh, was shown by compositional analyses, Fourier transform infrared (FTIR) spectroscopy, linkage analysis, and C-13-NMR spectroscopy to be a carrageenan composed predominantly of the repeating disaccharides 6'-O-methylcarrabiose 2,4'-disulfate, carrabiose 2,4-disulfate (the repeating unit of L-carrageenan), 4',6'-O-(1-carboxyethylidene)carrabiose 2-sulfate, and 6'-O-methylcarrabiose 2-sulfate. The carrageenan also contained small amounts of 4-linked Galp residues, some bearing methyl ether substitution at O-3 and some possibly bearing sulfate ester and/or glycosyl substitutions at O-3. The A. congesta carrageenan had unique rheological properties, its gels having some similarities to those of commercial iota -carrageenan but with the viscosity of commercial lambda -carrageenan. Polysaccharides from A. ligulata Harvey ex J. Agardh and A. stuartii Harvey were shown by constituent sugar and FTIR analyses to be sulfated galactans rich in mono-O-methylgalactose. The carrageenan structures of Areschougia spp. were consistent with those of the genera Rhabdonia, Erythroclonium, and Austroclonium, the other genera constituting the family Areschougiaceae.

A nearly idealized 6 '-O-methylated t-carrageenan from the Australian red alga Claviclonium ovatum (Acrotylaceae, Gigartinales)

The polysaccharides extracted from Claviclonium ovatum were studied by a combination of compositional assays, reductive partial hydrolysis, linkage analysis, Fourier Transform infrared (FTIR) spectroscopy, and C-13, H-1, and C-13/H-1 heteronuclear multiple quantum correlation (HMQC) two-dimensional nuclear magnetic resonance (NMR) spectroscopy. The chemical and spectroscopic data showed that the alkali-modified C. ovatum polysaccharides are composed of a nearly idealized repeating unit of 6'-O-methylcarrabiose 2,4'-disulfate (the repeating unit of 6-O-methylated iota-earrageenan), although some minor components were also present. The C. ovatum galactans are the most highly methylated carrageenans reported. (C) 2004 Elsevier Ltd. All rights reserved.

Anti-proliferative effects of novel Glyco-lipid-arsenicals (III) on MCF-7 human breast cancer cells

Arsenic trioxide appears to be effective in the treatment of pro-myelocytic leukaemia. The substituted phenylarsen(III)oxides are highly polar, they have a high tendency to undergo oxidation to As (V) and to form oligomers, to prevent this we protected the As-(OH)2 group as cyclic dithiaarsanes. To increase the compound's biological stability and passive diffusion we conjugated the compound of interest with lipoamino acids (Laas). Alternatively, we further conjugated the dithiaarsane derivative with a carbohydrate to utilize active transport systems and to target compound. We investigated two novel glyco-lipid arsenicals (III) (compounds 9 and 11) for their ability to initiate MCF-7 breast cancer cell death and characterized the mechanism by which death was initiated. A significant decrease in MCF-7 cell proliferation was observed using 1 μM and 10 μM compound (11) and 10 μM of compound (9). Treatment with compound (11) triggered apoptosis of MFC-7 cells while compound (9) induced inhibition of cellular proliferation was not via rapid induction of apoptosis and more likely reflected necrosis and/ or alterations in the cell cycle. Differences in the anti-proliferative potency of the two compounds indicate that structural modifications influence effectiveness. © 2006 Bentham Science Publishers Ltd.

Copper(II) complexes of mono- and di-nucleating hexaamines

The potentially hexadentate polyamines N,N,N',N'-tetrakis(2-aminoethyl)ethane-1,2-diamine (L-1) and the octamethylated analogue N,N,N',N'-tetrakis(2-dimethylaminoethyl)ethane 1,2-diamine (L-2) have been complexed with copper(II) and the crystal structures of their complexes determined. A trigonal-bipyramidal co-ordination geometry for [Cu(HL1)][ClO4](3) was found where one aminoethyl arm is not co-ordinated. By contrast, a dinuclear structure of formula [(H2O)Cu(L-2)Cu(OH)](3+) was determined for the N-methylated analogue, where the hexaamine acts as a bridging ligand between the two square-pyramidal metal centres. Electronic and EPR spectroscopy are both consistent with these structures being maintained in solution.

Carrageenans with complex substitution patterns from red algae of the genus Erythroclonium

Extracellular polysaccharides from three Erythroclonium spp. were shown, by a combination of compositional, linkage analyses, and Fourier transform infrared and C-13-nuclear magnetic resonance spectroscopy, to be highly substituted carrageenans with at least five types of repeating disaccharide units. These are the carrabiose 2,4'-disulfate of iota-carrageenan, carrabiose 2-sulfate of alpha-carrageenan, the 6'-O-methylated counterparts of each of these repeating units, and 4',6'-O-(1-carboxyethylidene)carrabiose 2-sulfate. The polysaccharides also contain significant amounts of unsubstituted, 4-linked galactopyranose and small amounts of 4-linked 3-O-methylgalactopyranose and terminal glycosyl residues. The carrageenan preparations of the three species are similar, differing only in the proportions of some components. (C) 1998 Elsevier Science Ltd.

Speciation of arsenic metabolites in the urine of occupational workers and experimental rats using an optimised hydride cold-trapping method

A hydride cold-trapping technique was developed and optimised for the measurement of urinary arsenic metabolites. The analytical precision of the method was found to be 6.1, 4.0 and 4.8% (n = 5) for inorganic arsenic (As-i), monomethylarsonate (MMA) and dimethylarsinate (DMA), respectively, with recoveries close to 100%, The detection limits were 1.0, 1.3 and 3 ng for As-i, MMA and DMA, respectively. The method was then used to analyse urine samples obtained from three groups of workers for occupational exposure in three companies where copper chrome arsenate was used for timber treatment. The results were compared with those for a normal control group of laboratory workers. Arsenic and its metabolites were also measured in experimental rats given 5 mg As kg(-1) body mass by oral gavage in the form of sodium arsenite, calcium arsenite or sodium arsenate. Occupational workers showed a significantly higher excretion of As-i, Up to two fold increases of urinary As-i excretion in rats compared with control rats were also observed in animals dosed with various forms of arsenicals. The method is suitable for the measurement of arsenic metabolites in urine of both humans and experimental animals.

N-methylation of macrocyclic hexaaminecobalt(III) complexes

Reaction between formaldehyde and the pendant arm macrocyclic complex (trans-6,13-dimethyl-1,4,8,11-tetraazacyclotetradecane-6,13-diamine)cobalt(III) [CoL1](3+) yielded the diimine derivative trans-6,13-dimethyl-6.13-bis(methyleneamino)-1,4,8,11-tetraazacyclotetradecane (L-3) as its cobalt(III) complex. Reduction of the imines has been achieved with NaBH4 and the meso and rac cobalt(III) complexes of trans-6,13-dimethyl-6,13-bis(methylamino)-1,4,8,11-tetraazacyclotetradecane (L-5) have been prepared. Crystal structures of the macrocyclic complexes [CoL1][ClO4](3), [CoL3][ClO4](3) and meso-[CoL5][ClO4](3).2H(2)O were determined and some unusual structural, spectroscopic and electrochemical variations observed going from the parent hexaamine [CoL1](3+) to [CoL3](3+) (diimine) and ultimately to [CoL5](3+) (bis-N-methylated hexaamine).

Carrageenans from Australian representatives of the family Cystocloniaceae (Gigartinales, Rhodophyta), with description of Calliblepharis celatospora sp. nov., and transfer of Austroclonium to the family Areschougiaceae

Ten Australian representatives from seven of the 10 genera presently constituting the family Cystolcloniaceae have been analyzed for their cell-wall galactans. Included in our survey are the monotypic Australian-endemic genera Austroclonium, Gloiophyllis, Erythronaema, and Stictosporum, one species of Craspedocarpus, three species of Rhodophyllis, and two species of Calliblepharis. As one of the species of the latter genus is endemic to Western Australia and presently undescribed, we illustrate its habit and anatomical features in formally proposing to name it Calliblepharis celatospora Kraft, sp. nov. All the species surveyed essentially produce typical iota (iota)-carrageenans, with the exception of Austroclonium. The sulfated galactans from Austroclonium predominantly contain the repeating units of iota-, alpha (alpha)-, and 6'-O-methylated iota- and alpha-carrageenans; whether these exist as discrete polysaccharides or a complex hybrid structure was not resolved. Thus, Austroclonium carrageenans resemble the polysaccharides from Rhabdonia, Areschougia, and Erythroclonium. Although these latter three genera are currently included in the large gigartinalean family Solieriaceae, all produce significantly different carrageenans from Solieria itself and related genera such as Eucheuma, Kappaphycus, Betaphycus, Sarcodiotheca, Agardhiella, Sarconema, and Callophycus. In consideration of these findings, as well as of significant anatomical similarities, we provisionally recommend reestablishment of the family Rhabdoniaceae Kylin (as the family Areschougiaceae J. Agardh) for Rhabdonia, Areschougia, Erythroclonium, and Austroclonium.

Nuapapuin A and sigmosceptrellins D and E: New norterpene cyclic peroxides from a southern Australian marine sponge, Sigmosceptrella sp.

A Sigmosceptrella sp. from the Great Australian Eight, Australia, has yielded the new norditerpene cyclic peroxide, nuapapuin A (2a), and the norsesterterpene cyclic peroxide sigmosceptrellin D (3a), characterized as the corresponding methyl esters 2b and 3b. The crude methylated sponge extract also yielded the new norsesterterpene cyclic peroxide sigmosceptrellin E methyl ester (4). Relative stereochemistry about C2, C3, and C6 was assigned by established empirical rules and absolute stereochemistry by the advanced Mosher procedure. A plausible biosynthetic pathway has been proposed that rationalizes key transformations in the biosynthesis of all known norterpene cyclic peroxides and related norterpene ketones, dienes and sigmosceptrins.

Antigen-specific suppression of inflammatory arthritis by dendritic cells

Purpose Antigen-specific suppression of a previously primed immune response is a major challenge for immunotherapy of autoimmune disease. We have shown that NF-κB inactivation in dendritic cells (modified DC) converts them into cells that tolerize rather than immunize to specific antigen [1]. Antigen-exposed modified DC prevent priming of immunity, and they suppress previously primed immune responses. Regulatory CD4+ T cells, which can transfer antigen-specific tolerance in an IL-10-dependent fashion, mediate the tolerance. We hypothesized that modified DC exposed to arthritogenic antigen would suppress clinical arthritis after disease onset. Methods Antigen-induced arthritis was induced in C57/Bl6 mice by priming to methylated bovine serum albumin (mBSA) antigen followed by challenge injection of mBSA to one knee. Knee swelling was apparent within 2 days, with peak clinical signs apparent at 5 days. Mice were treated with antigen-exposed modified DC between 2 and 6 days after mBSA challenge to the knee joint. Results Clinical arthritis was suppressed in each group receiving mBSA-exposed modified DC within 4 days compared with mice that received either no DC or keyhole limpet hemocyanin-exposed modified DC. Clinical improvement was associated with mBSA-specific tolerance in mice receiving mBSA-exposed modified DC. Tolerance induction was not impaired by concomitant administration of anti-tumor necrosis factor alpha monoclonal antibody. Subsequent rechallenge with intra-articular IL-1 induced flare of arthritis in all groups, which could be effectively suppressed by a second administration of mBSA-exposed modified DC. Conclusions The data indicate that modified DC induce antigen-specific immune suppression in this model of inflammatory arthritis, even after full clinical expression of the disease. These observations have important implications for antigen-specific therapy of autoimmunity.

Folate-sensitive fragile site FRA10A is due to an expansion of a CGG repeat in a novel gene, FRA10AC1, encoding a nuclear protein

Fragile sites appear visually as nonstaining gaps on chromosomes that are inducible by specific cell culture conditions. Expansion of CGG/ CCG repeats has been shown to be the molecular basis of all five folate-sensitive fragile sites characterized molecularly so far, i.e., FRAXA, FRAXE, FRAXF, FRA11B, and FRA16A. In the present study we have refined the localization of the FRA10A folate-sensitive fragile site by fluorescence in situ hybridization. Sequence analysis of a BAC clone spanning FRA10A identified a single, imperfect, but polymorphic CGG repeat that is part of a CpG island in the 5'UTR of a novel gene named FRA10ACl. The number of CGG repeats varied in the population from 8 to 13. Expansions exceeding 200 repeat units were methylated in all FRA10A fragile site carriers tested. The FRA10ACl gene consists of 19 exons and is transcribed in the centromeric direction from the FRA10A repeat. The major transcript of similar to 1450 nt is ubiquitously expressed and codes for a highly conserved protein, FRA10ACl, of unknown function. Several splice variants leading to alternative 3' ends were identified (particularly in testis). These give rise to FRA10ACl proteins with altered COOH-termini. Immunofluorescence analysis of full-length, recombinant EGFP-tagged FRA10ACl protein showed that it was present exclusively in the nucleoplasm. We show that the expression of FRA10A, in parallel to the other cloned folate-sensitive fragile sites, is caused by an expansion and subsequent methylation of an unstable CGG trinucleotide repeat. Taking advantage of three cSNPs within the FRA10ACl gene we demonstrate that one allele of the gene is not transcribed in a FRA10A carrier. Our data also suggest that in the heterozygous state FRA10A is likely a benign folate-sensitive fragile site. (C) 2004 Elsevier Inc. All rights reserved.