Presence Of Beta-glucuronidase Research Articles

Benzyl Ether-Linked Glucuronide Derivative of 10-Hydroxycamptothecin Designed for Selective Camptothecin-Based Anticancer Therapy

A beta-glucuronidase-activated prodrug approach was applied to 10-hydroxycamptothecin, a Camptotheca alkaloid with promising antitumor activity but poor water solubility. We synthesized a glucuronide prodrug of 10-hydroxycamptothecin ( 7) in which glucuronic acid is connected via a self-immolative 3-nitrobenzyl ether linker to the 10-OH group of 10-hydroxycamptothecin. Compound 7 was 80 times more soluble than 10-hydroxycamptothecin in aqueous solution at pH 4.0 and was stable in human plasma. Prodrug 7 was 10- to 15-fold less toxic than the parent drug to four human tumor cell lines. In the presence of beta-glucuronidase, prodrug 7 could be activated to elicit similar cytotoxicity to the parent drug in tumor cells. Enzyme kinetic studies showed that Escherichia coli beta-glucuronidase had a quite low K m of 0.18 microM for compound 7 and exhibited 520 times higher catalytic efficiency for 7 than for 6 (a glucuronide prodrug of 9-aminocamptothecin). Molecular modeling studies predicted that compound 7 would have a higher binding affinity to human beta-glucuronidase than compound 6. Prodrug 7 may be useful for selective cancer chemotherapy by a prodrug monotherapy (PMT) or antibody-directed enzyme prodrug therapy (ADEPT) strategy.

Corynebacterium seminale sp. nov., a new species associated with genital infections in male patients.

We studied 12 coryneform isolates having similar biochemical profiles which did not permit their assignment to any recognized taxa. Human semen was the source for seven of these strains, whereas the other strains were isolated from urethra, urine, and blood specimens of adult male patients. These bacteria were found in significant quantities (10(4) to 10(5) CFU/ml) in semen specimens from infertile male patients with the diagnosis of prostatitis. These strains had characteristics of the genus Corynebacterium, such as 60 mol% G + C in the DNA and corynemycolic acids, meso-diaminopimelic acid, arabinose, and galactose in the cell wall. Quantitative DNA-DNA hybridizations (S1 nuclease procedure) and phylogenies based on comparisons of almost-complete small-subunit ribosomal DNA sequences confirmed that these strains constitute a single new species within the genus Corynebacterium. All 12 strains showed similar phenotypic features, i.e., good growth on sheep blood agar in contrast with poor growth on the same medium supplemented with 1% Tween 80, a positive CAMP test in the presence of Staphylococcus aureus, glucose and sucrose fermentation, and the presence of beta-glucuronidase. Some strains reduced nitrate and hydrolyzed urea or esculin. These features allowed us to distinguish these strains from members of any other coryneform taxon, and the proposed name is Corynebacterium seminale with strain IBS B12915 (CIP 104297) as the type strain. The description and delineation of these strains as a new species should be useful for further studies, including evaluations of their prevalence among the normal flora and their clinical implications.

Sequential degradation of chondroitin sulfate in molluscs. Desulfation of chondroitin sulfate without prior depolymerization by a novel sulfatase from Anomalocardia brasiliana

A sulfatase acting upon chondroitin sulfate polymers, free of beta-glucuronidase and beta-N-acetylhexosaminidases, was isolated from extracts of the mollusc Anomalocardia brasiliana. The enzyme totally desulfates both chondroitin 4- and 6-sulfates without concomitant depolymerization of the compounds. It has no activity upon heparan sulfate, heparin, dermatan sulfate, and chondroitin sulfate disaccharides. It shows a pH of 5.0 and a temperature of 37 degrees C for optimum activity with a Km of 4 x 10(-5) M. The sulfatase is inhibited by sulfate and phosphate ions and HgCl2. The latter inhibition is reverted by sodium tetrathionate. Contrary to the sulfatases described so far the enzyme is activated by the lactone of D-saccharic acid when in the presence of beta-glucuronidase and beta-N-acetylgalactosaminidase. Several experiments indicate that the sulfatase is the first enzyme in the sequential degradation of chondroitin sulfate in the mollusc. This differs from the pathway of degradation of this compound in vertebrates and bacteria.

Effect of Microsomal Enzyme Inducers on the Urinary Excretion Pattern of Mutagenic Metabolites of the Carcinogen 2,4-Toluenediamine

2,4-Toluenediamine [(TDA) CAS: 95-80-7] was administered to rats pretreated with the microsomal enzyme inducers phenobarbital (PB), beta-naphthoflavone (beta NF), or 3-methylcholanthrene (MCA). The 24-hour urines of male F344 rats were examined for their mutagenic potency by means of the Salmonella assay, with the Aroclor 1254-pretreated rat liver S-9 fraction as an activating system. No revertants were found with TDA or its urinary metabolites in the absence of the S-9 fraction. In the presence of S-9, the number of revertants increased as the concentration of TDA or its urinary metabolites increased. The urinary metabolites, generated after the microsomal enzyme inducers (PB, beta NF, MCA), had increased mutagenic activity as compared with the controls (saline, corn oil). In the presence of beta-glucuronidase (beta G), increased numbers of TA98 revertants were noted in the urine of rats pretreated with PB, saline, or corn oil. Addition of sulfatase did not alter the number of TA98 revertants. Conversely, beta G treatment of urine from rats pretreated with MCA or beta NF led to a decrease in the number of TA98 revertants as compared to levels in urine without beta G. Addition of known urinary metabolites of TDA, such as 4-acetylamino-2-aminobenzoic acid or 2,4-diacetylaminobenzoic acid, to beta NF-pretreated rat urine had no inhibitory effect on the mutagenicity in the absence of beta G. However, in the presence of beta G, the inhibitory effect was similar to that noted with beta NF-pretreated rat urine. Upon separation of urinary metabolites (beta NF-pretreated rat urine) into free, conjugated, and water-soluble forms, the maximum number of TA98 revertants was associated with the free ethyl acetate-extractable fraction, which accounted for the total mutagenic activity associated with the original volume of urine. Conjugated metabolites showed much less mutagenic activity, and an inhibitory principle was associated with the water-soluble fraction.

Assay for Mutagenicity of Bile in Sprague-Dawley Rats Treated Subcutaneously With Intestinal Carcinogens

To investigate the mode of action of sc injected intestinal carcinogens, the mutagenicity assay of bile collected from noninbred Sprague-Dawley rats treated sc with carcinogens was conducted in the presence and absence of beta-glucuronidase. The bile samples from rats inoculated with 4-aminobiphenyl were mutagenic for Salmonella typhimurium TA100 only in the presence of beta-glucuronidase, whereas those from the 3,2'-dimethyl-4-aminobiphenyl-treated rats did not require the enzyme for mutagenicity toward strain TA100. On the contrary, the assays with S. typhimurium G46 and TA100 of bile from rats inoculated with 1,2-dimethylhydrazine, azoxymethane, or methylazoxymethanol acetate failed to reveal mutagenicity whether beta-glucuronidase was added or not, though these carcinogens were highly mutagenic for strain G46 in the Salmonella-microsome mutagenicity test and/or in the host-mediated assay.

Conjugated Catechol Derivatives in a Transplantable Islet Cell Tumour of the Golden Hamster

Two glucuronidated catechol have been identified in a transplantable islet cell tumour of the golden hamster, i.e. dopamine-4-O-glucuronide and 3-methoxytyramine-4-O-glucuronide. L-dopa is rapidly metabolized in the tumour to one or both of these glucuronides. Incubation of tumour homogenates in the presence of beta-glucuronidase shows that dopamine-4-O-glucuronide is present in the tumour in extremely high concentrations.

Screening Plate Method for Detection of Bacterial β-Glucuronidase

For screening tests of bacterial beta-glucuronidase (EC 3.2.1.31) activity an agar plate (MUG plate) containing the enzyme substrate, 4-methylumbelliferyl-beta-D-glucuronide, was developed. In the presence of beta-glucuronidase, the strongly fluorescent 4-methylumbelliferone, which can be detected by ultraviolet light, was liberated. For tests of fastidious bacteria, the MUG-plate was enriched with supplements containing heat-labile growth factors without influencing the reaction. The plate was found to be suitable for screening tests. The sensitivity of the MUG-plate was determined by means of fluorimetric method. With the present method, beta-glucuronidase activities were demonstrated in some well-known beta-glucuronidae-producing microorganisms as well as in Bacteroides and Corynebacterium spp.