Mutagenic activation of biliary metabolites of 1-nitropyrene by intestinal microflora

To investigate the modifying role of intestinal microflora in the metabolism of chemical carcinogens in vivo, we subjected bile from Fischer rats treated per os with chemical carcinogens and related compounds to a mutagenicity assay in the presence and absence of a cell-free extract from human feces. A mixture of the bile sample and potassium phosphate buffer was incubated in the presence or absence of human cell-free fecal extract and then further incubated with a bacterial suspension of Salmonella typhimurium tester strains TA98 or TA100. Bile from rats treated with 1-nitropyrene (1-NP) produced about 2700 and 400 revertants per plate in strain TA98 in the presence and absence of the fecal extract, respectively. There was a drug dose- and bile volume-related response. Treatment of 1-NP-bile with beta-glucuronidase, but not aryl sulfatase, enhanced its mutagenicity. Cell-free extracts of some strains of intestinal bacteria (Bacteroides fragilis ATCC 12044, B. vulgatus ATCC 8482, B. thetaiotaomicron ATCC 12290, Bacteroides sp. strain 524, Eubacterium eligens VPI C15-48, Peptostreptococcus sp. strain 204 and Escherichia coli A-5-18) also enhanced the mutagenicity of 1-NP-bile. These bacterial cell-free extracts hydrolyzed the synthetic beta-D-glucuronides of phenolphthalein and/or p-nitrophenol. These data indicate that the glucuronide(s) of 1-NP-metabolite(s) secreted into bile can be hydrolyzed in the intestine by bacterial beta-glucuronidases to potent mutagenic aglycone(s).     

Effect of sulfur supply on sulfate uptake,and alkaline sulfatase activity in free-living and symbiotic bradyrhizobia

The effect of sulfur limitation on sulfate transport and metabolism was studied in four bradyrhizobia strains using sulfur-limited and sulfur-excess chemostat cultures. Characteristics of bradyrhizobia associated with sulfurlimitation were determined and these parameters used to bioassay the sulfur status of bacteroids in nodules on sulfur adequate or sulfur deficient soybean and peanut plants. Sulfur-limited cells took up sulfate 16- to 100-fold faster than sulfur-rich cells. The sulfate-uptake system appeared similar in all strains with apparent K _m values ranging from 3.1 M to 20 M sulfate with maximum activities between 1.6 and 10 nmol·min^-1·mg^-1 protein of cells. Sulfate-limited cells of all strains derepressed the enzyme alkaline sulfatase in parallel with the derepression of the sulfate transport system. Similarly, the initial enzyme of sulfate assimilation (ATP sulfurylase) was fully derepressed in sulfur-limited cultures. Bacteroids isolated from sulfur adequate and sulfur deficient soybean and peanut possessed very limited sulfate uptake activity and low levels of activity of ATP sulfurylase as well as lacking alkaline sulfatase activity. These results indicate bacteriods have access to adequate sulfur to meet their requirements even when the host plant is sulfur-deficient.Abbreviations CCCP Carbonyl cyanide m-chlorophenylhydrazone - DCCD N,N-dicyclohexyl carbodiimide     

A radioimmunoassay method for urinary catechol estrogens

A radioimmunoassay method for urinary catechol estrogens is described. The specific nature of the antisera allows direct analyses of acid hydrolyzed urine. A LH-20 Sephadex column chromatography can be employed for individual determinations of 2-hydroxyestrone and 2-hydroxyestradiol. The excretion of catechol estrogens during menstrual cycles ranged from 14.48 to 50.15 μg per 24 hours, whereas, during the last trimester of pregnancies, the values ranged from 129.30 to 1758.20 μg per 24 hours.     

Evidence for the Cd<Superscript>2+</Superscript> Activation of the Aryl Sulfatase from <Emphasis Type="Italic">Helix pomatia</Emphasis>

Often used to remove sulfate groups from carbohydrates, the regulatory properties of the aryl sulfatase from Helix pomatia remain little characterized. As many hydrolytic enzymes utilize exogenous metal ions in catalysis, the effect of various divalent metal ions on the sulfatase was investigated. Evidence for metal ion activation was collected, with Cd²⁺ being notable for effective activation. The enzyme was inhibited by Cu²⁺. The response of other common hydrolases to divalent metal ions was characterized. Activation by Cd²⁺ was not observed for chymotrypsin, rabbit liver esterase, or β-galactosidase. Instead, Cd was found to inhibit both the esterase and the galactosidase. Inhibition by Cu²⁺ and Zn²⁺ was also observed for some of these hydrolases.     

CONCENTRATIONS OF ESTRONE SULFATE and PROGESTERONE IN PLASMA and SALIVA, VAGINAL CYTOLOGY, and BIOELECTRIC IMPEDANCE DURING THE ESTROUS CYCLE OF THE HAWAIIAN MONK SEAL (MONACHUS SCHAUNSLAnDI)

Blood and saliva samples, vaginal swabs, and bioelectric impedance measurements were collected 1–3 times per week from a captive adult female Hawaiian monk seal ( Monacbus schauinslandi ) during the spring and summer of 1991. During 1992 saliva only was collected on average 4 times weekly. Concentrations of progesterone and estrone sulfate, and the periodic appearance of cornified epithelial cells from the vagina indicated consecutive estrous cycles ranging 35 ± 3 days in duration. Progesterone concentrations in plasma and saliva had a correlation of 0.868, while estrone sulfate had a correlation of 0.982. Bioimpedance of the whole seal body resulted in a pattern similar to the estrone sulfate concentrations, but shifted forward by 2 d. Bioimpedance of the upper vaginal tissues paralleled the estrone sulfate pattern while the bioimpedance of the lower reproductive tract fluctuated without a distinct pattern. The luteal phase ranged 17–20 d and the follicular phase was 15–18 d in length. These results indicate that: (1) the Hawaiian monk seal, in captivity, is a polyestrous seal, in contrast to other phocid seals that have been studied; (2) salivary concentrations of estrogen and progesterone may provide an accurate, less-invasive method of monitoring reproductive hormones in captive Hawaiian monk seals; and (3) vaginal cytology and bioelectric impedance reflect physiological changes associated with the estrous cycle.     

Structural and Functional Characterization of the BcsG Subunit of the Cellulose Synthase in Salmonella typhimurium

Many bacteria secrete cellulose, which forms the structural basis for bacterial multicellular aggregates, termed biofilms. The cellulose synthase complex of Salmonella typhimurium consists of the catalytic subunits BcsA and BcsB and several auxiliary subunits that are encoded by two divergently transcribed operons, bcsRQABZC and bcsEFG. Expression of the bcsEFG operon is required for full-scale cellulose production, but the functions of its products are not fully understood. This work aimed to characterize the BcsG subunit of the cellulose synthase, which consists of an N-terminal transmembrane fragment and a C-terminal domain in the periplasm. Deletion of the bcsG gene substantially decreased the total amount of BcsA and cellulose production. BcsA levels were partially restored by the expression of the transmembrane segment, whereas restoration of cellulose production required the presence of the C-terminal periplasmic domain and its characteristic metal-binding residues. The high-resolution crystal structure of the periplasmic domain characterized BcsG as a member of the alkaline phosphatase/sulfatase superfamily of metalloenzymes, containing a conserved Zn²⁺-binding site. Sequence and structural comparisons showed that BcsG belongs to a specific family within alkaline phosphatase-like enzymes, which includes bacterial Zn²⁺-dependent lipopolysaccharide phosphoethanolamine transferases such as MCR-1 (colistin resistance protein), EptA, and EptC and the Mn²⁺-dependent lipoteichoic acid synthase (phosphoglycerol transferase) LtaS. These enzymes use the phospholipids phosphatidylethanolamine and phosphatidylglycerol, respectively, as substrates. These data are consistent with the recently discovered phosphoethanolamine modification of cellulose by BcsG and show that its membrane-bound and periplasmic parts play distinct roles in the assembly of the functional cellulose synthase and cellulose production.     

The development and application of an enzyme immunoassay for urinary estrone conjugates

An enzyme immunoassay (EIA) for estrone conjugates is described and applied to urine samples from a female Indian rhinoceros, a female gorilla, and a female lion-tailed macaque. Concomitant measures of estrone conjugates in the same sample are compared to the values obtained with radioimmunoassay. High correlation coefficients for values obtained from each assay indicate that EIA measurements provide information that is comparable to values obtained by radioimmunoassay. EIA methods for urinary steroid conjugates can provide a practical tool to evaluate female reproductive status of zoo species without the need for a traditional endocrine laboratory.     

Menstrual cycle characterization and artificial insemination in the black mangabey (Cercocebus aterrimus)

Concentrations of immunoreactive estrone conjugates, pregnanediol-3-glucuronide, and luteinizing hormone were measured and indexed to creatinine in daily urine samples from three female black mangabeys (Cercocebus aterrimus). Daily observations of menstruation and perineal tumescence were recorded. The mean ± SEM lengths of the menstrual cycle [apparent cycle length of 26.0 ± 0.8 days determined by observation of intermenstrual intervals (n = 26); physiologic cycle length of 31.3 ± 5 days determined by urinary endocrine analysis (n = 4)], follicular phase [16.5 ± 4 days (n = 4)], and luteal phase [14.8 ± 1 day (n = 4)] were determined. The apparent cycle length is probably more accurate. Perineal tumescence began during or shortly after menstruation, increased concomitantly with increasing follicular phase conjugated estrone values, and reached maximal size in the periovulatory period. Ovulation was closely followed by a drop in conjugated estrone levels, an increase in urinary pregnanediol-3-glucuronide, and perineal detumescence. Peak concentrations of conjugated estrone and luteinizing hormone values were coincident. Pregnanediol-3-glucuronide accurately reflected luteal function in the black mangabey. Knowledge of the menstrual cycle parameters and their correlation to perineal tumescence was used to time artificial inseminations. Semen was obtained by rectal electroejaculation. Coagulum and extended semen, or trypsin-digested coagulum, were used for insemination. One insemination of trypsin-digested coagulum at the external os of the cervix resulted in a probable conception, follówed by apparent abortion after 3 weeks.     

Role of cholesterol sulfate in epidermal structure and function: Lessons from X-linked ichthyosis

X-linked ichthyosis is a relatively common syndromic form of ichthyosis most often due to deletions in the gene encoding the microsomal enzyme, steroid sulfatase, located on the short area of the X chromosome. Syndromic features are mild or unapparent unless contiguous genes are affected. In normal epidermis, cholesterol sulfate is generated by cholesterol sulfotransferase (SULT2B1b), but desulfated in the outer epidermis, together forming a ‘cholesterol sulfate cycle’ that potently regulates epidermal differentiation, barrier function and desquamation. In XLI, cholesterol sulfate levels my exceed 10% of total lipid mass (≈ 1% of total weight). Multiple cellular and biochemical processes contribute to the pathogenesis of the barrier abnormality and scaling phenotype in XLI. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.     

Src supports UDP-glucuronosyltransferase-2B7 detoxification of catechol estrogens associated with breast cancer

Mammary gland-distributed and ER-bound UDP-glucuronosyltransferase (UGT)-2B7 metabolizes genotoxic catechol-estrogens (CE) associated with breast cancer initiation. Although UGT2B7 has 3 PKC- and 2 tyrosine kinase (TK)-sites, its inhibition by genistein, herbimycin-A and PP2 with parallel losses in phospho-tyrosine and phospho-Y438-2B7 content indicated it requires tyrosine phosphorylation, unlike required PKC phosphorylation of UGT1A isozymes. 2B7 mutants at PKC-sites had essentially normal activity, while its TK-sites mutants, Y236F- and Y438F-2B7, were essentially inactive. Overexpression of regular or active Src, but not dominant-negative Src, in 2B7-transfected COS-1 cells increased 2B7 activity and phospho-Y438-2B7 by 50%. Co-localization of 2B7 and regular SrcTK in COS-1 cells that was dissociated by pretreatment with Src-specific PP2-inhibitor provided strong evidence Src supports 2B7 activity. Consistent with these findings, evidence indicates an appropriate set of ER proteins with Src-homology binding-domains, including 2B7 and well-known multi-functional Src-engaged AKAP12 scaffold, supports Src-dependent phosphorylation of CE-metabolizing 2B7 enabling it to function as a tumor suppressor.