Arylsulphatases in human brain: separation, purification, and certain properties of the two soluble arylsulphatases

Abstract— Arylsulphatases (aryl-sulphate sulphohydrolases; E.C. 3.1.6.1) in the soluble subcellular fraction (105000g, 2 h) of human brain were partially purified by ammonium sulphate fractionation, ion exchange chromatography, and Sephadex gel filtration. Potassium-4-methylumbelliferone-sulphatase (MUS-sulphatase) adsorbed on DEAE-cellulose was purified approximately 700-fold over activity in the soluble fraction and the unadsorbed MUS-sulphatase was similarly purified approximately 600-fold. The arylsulphatase adsorbed to DEAE-cellulose exhibited a K_m value for MUS of 12.5 mM and a pH optimum of 5.7, whereas the unadsorbed arylsulphatase exhibited a K_m value for MUS of 8.3 mM and a pH optimum of 5.4. The molecular weights of the two enzymes were approximately 109,600 and 51,300, respectively. Sulphate (0.5 mM) showed pronounced mixed inhibition only of the unadsorbed arylsulphatase. Ag⁺ ions (0.25 mM) showed 96 per cent inhibition of the adsorbed arylsulphatase, whereas an activation of the unadsorbed arylsulphatase was observed.     

Automated analysis of the pyridinium crosslinks of collagen in tissue and urine using solid-phase extraction and reversed-phase high-performance liquid chromatography.

A fully automated method for assaying the collagen crosslinking amino acids, pyridinoline and deoxypyridinoline, in human urine samples or tissue hydrolysates is described. Samples were processed using a Gilson ASPEC system with solid-phase extraction of the crosslinks on columns containing 100 mg of microgranular cellulose. Introduction of an additional solvent step during sample preparation allowed direct analysis by reversed-phase HPLC and elimination of the drying step used previously in a manual method. Use of a synthetic pyridinoline derivative as internal standard enabled accurate quantification of the crosslinks by correcting for recoveries through the whole assay. Samples were analyzed in sequential mode with a total assay time of 30 min. The automated assay showed close correlation with the manual method for both free and total crosslink determinations in human urine (r > 0.97). Reproducibility was improved, as seen from replicate analyses of human urine (CV < 3% for automated pyridinoline measurement compared with 8-12% previously observed for the manual method). Crosslink excretion is the most useful marker of collagen degradation in metabolic bone diseases and arthritic disorders. The automated assay which has been developed is rapid, convenient, and reliable and will greatly facilitate the monitoring of urinary collagen crosslinks and their tissue levels in clinical investigations.     

Effect of exogenous DNA fragments on human cell extract-mediated DNA repair synthesis

Extracts from HeLa cells were used to study the susceptibility of repair synthesis in UV-irradiated plasmid DNA to inhibition by exogenously added nucleic acid. Purified DNA restriction fragments have little inhibitory effect on repair synthesis. However, activated calf thymus DNA fragments, genomic DNA fragments in cell extracts, and sonicated plasmid DNA all inhibited repair synthesis. Degraded DNA fragments arising from E. coli during bacterial plasmid purification were found to be particularly inhibitory. tRNA is not a potent inhibitor of in vitro repair synthesis. In order to observe efficient DNA repair synthesis mediated by human cell extracts, it is essential to prepare highly purified closed circular plasmid DNA, and we describe a reliable method for doing so.     

Toxicity of quinoline alkaloids to cultured Cinchona ledgeriana cells

The toxicity of Cinchona alkaloids to cell cultures of C. ledgeriana has been studied in relation to alkaloid uptake and possibilities for selecting high-yielding cell lines. The most toxic, quinine, was completely toxic at 5.5 mM. Both quinine and quinidine were more toxic than their unmethoxylated precursors, cinchonidine and cinchonine. The permanently-charged metho-chlorides of quinine and cinchonidine were less toxic than the parent alkaloids, despite showing similar accumulation ratios in 5-day uptake experiments at sub-toxic concentrations (ca 1.7mM). The toxicity of the natural quinoline alkaloids appears to be a non-specific effect which may be caused by intracellular alkalinisation following uptake of the uncharged bases. The use of precursors of quinine and quinidine as toxic agents for the selection of cell lines with enhanced quinine and quinidine production is ruled out by the lower toxicity of these precursors and by the correlation of an apparently non-specific toxicity with uptake.     

Potential for use of nicotinic acid as a selective agent for isolation of high nicotine-producing lines of Nicotiana rustica hairy root cultures

The addition of exogenous nicotinic acid, nicotinamide or nicotine was studied with reference to their effects on growth and alkaloid production by hairy root cultures of Nicotiana rustica. Nicotinic acid and nicotinamide were toxic (50% phytostatic dose being 2.4 and 9 mM respectively) while nicotine was not toxic below 10 mM. Nicotinic acid (up to 5 mM) was found to be phytostatic rather than phytotoxic. Roots exposed to increasing nicotinic acid or nicotinamide levels had altered alkaloid accumulation patterns relative to the controls. The principal effects were to increase the intracellular and extracellular levels of anatabine and nicotine, with a markedly greater proportion of anatabine being produced. The use of nicotinic acid as a selection agent for the recovery of higher alkaloid-producing lines is identified and discussed.     

Complementation of the xeroderma pigmentosum DNA repair defect in cell-free extracts

Soluble extracts from human lymphoid cell lines that perform repair synthesis on covalently closed circular DNA containing pyrimidine dimers or psoralen adducts are described. Short patches of nucleotides are introduced by excision repair of damaged DNA in an ATP-dependent reaction. Extracts from xeroderma pigmentosum cell lines fail to act on damaged circular DNA, but are proficient in repair synthesis of ultraviolet-irradiated DNA containing incisions generated by Micrococcus luteus pyrimidine dimer-DNA glycosylase. Repair is defective in extracts from all xeroderma pigmentosum cell lines investigated, representing the genetic complementation groups A, B, C, D, H, and V. Mixing of cell extracts of group A and C origin leads to reconstitution of the DNA repair activity.     

Ribosomal gene reiteration in a marsupial species with an X-linked nucleolar organizer

The number of ribosomal cistrons in somatic cells of males and females of Macropus eugenii have been estimated using RNA/DNA hybridization in 70% (v/v) formamide. The male has one X-linked active nucleolar organizer and the female two and while the number of ribosomal cistrons is variable between the four males examined (31-65 copies), it is substantially less than the number present in the three females (94-103 copies). There is, therefore, no evidence of dosage compensation by amplification.     

Effect of dicarboxylic (C6 and C9) acids on a human squamous carcinoma cell line in culture

In tissue culture, azelaic acid (C9) has been shown to have an anti-proliferative and cytotoxic effect on human and murine malignant melanocytes, with inhibition of mitochondrial oxido-reductase enzymes and DNA synthesis, and damage to mitochondria. Recent reports of effects on differentiation of normal keratocytes have led to the present study of its effects on a squamous carcinoma cell line. Cells were exposed to single doses of disodium salts of azelaic (C9(2)Na) and adipic (C6(2)Na) acids at concentrations of 10(-2)M and 5 x 10(-2)M for 48 hrs. Only C9(2)Na at 5 x 10(-2) M for 4 hrs., and longer, significantly affected proliferation, and the cells exhibited massive swelling of mitochondria with loss of cristae. The results further confirm the probable value of azelaic acid as a general anti-tumoral agent rather than a specifically melanocytotoxic one. They could justify clinical studies on the effect of topical azelaic acid therapy on squamous cell carcinoma in vivo.