Bacterial expression and characterization of a cDNA for human liver estrogen sulfotransferase

A distinct human estrogen sulfotransferase (hEST-1) cDNA has been isolated from a human liver lambda Zap cDNA library using a PCR procedure. The enzymatically active protein has been expressed in two bacterial expression systems and the kinetic and immunologic properties of the enzyme have been characterized. The full-length cDNA for hEST-1 is 994 base pairs in length and encodes a 294 amino acid protein with a calculated molecular mass of 35,123 Da. Purified hEST-1 migrated with an apparent molecular mass of 35,000 Da during SDS-polyacrylamide gel electrophoresis. Immunoblot analysis of hEST-1 expressed in E. coli with a rabbit anti-hEST-1 antibody yields a band of approximately 35,000 Da. The anti-hEST-1 antibody also detects a single band in human liver and jejunum cytosol which migrates with the same molecular mass as expressed hEST-1. There was also no cross-reactivity of hEST-1 with rabbit anti-hP-PST or rabbit anti-hDHEA-ST antibodies upon immunoblot analysis. hEST-1 was expressed in bacteria and purified to homogeneity. Expressed hEST-1 activity has a significantly greater affinity for estrogen sulfation than that found for the other human STs which conjugate estrogens. hEST-1 maximally sulfates beta-estradiol and estrone at concentrations of 20 nM. hEST-1 also sulfates dehydroepiandrosterone, pregnenolone, ethinylestradiol, and 1-naphthol, at significantly higher concentrations; however, cortisol, testosterone and dopamine are not sulfated. The results presented in this paper describe the expression and characterization of a human EST distinct from other human STs which sulfate estrogens. The high affinity of hEST-1 for estrogens indicates that this ST may be important in both the metabolism of estrogens and in the regulation of their activities.     

Bioconcentration of xenobiotics in trout bile: a proposed monitoring aid for some waterborne chemicals

A technique is proposed for the monitoring of certain xenobiotic pollutants in suspect aquatic enviornments by fish bile analysis. Bile removed from rainbow trout (Salmo gairdneri) exposed to nine different radioactive compounds in vivo contained concentrations of radioactivity greater than those in the surrounding water. Bile-to-water radioactivity ratios as high as 10,000: 1 were found after 24-hour exposures. The results of these experiments suggest that analysis of bile of wild or caged fish from a suspect site may be useful as a qualitative monitoring aid for certain types of xenobiotics in water.     

Prolonged disease-free survival in bronchogenic carcinoma associated with HLA-Aw19 and HLA-B5. A two-year prospective study

Seventy patients with squamous carcinoma or adenocarcinoma of the lung were HLA typed at the time of their diagnosis and initial therapy. No abnormal HLA antigen frequencies were found. However, the possession of HLA-Aw19 or HLA-B5 was significantly correlated with two year disease-free survival. Twelve of 21 patients (57%) with either Aw 19 or B5 were disease-free at two years. In contrast, only six of 48 patients (13%) not having either antigen were disease-free (p less than .001). One patient was lost to follow-up. It is possible that Aw 19 and/or B5 confer resistance to progression of bronchogenic carcinoma on their possessors.     

Purification and characterization of an interleukin-1beta-converting enzyme family protease that activates cysteine protease P32 (CPP32)

CPP32, a member of the interleukin-1beta-converting enzyme (ICE) family of cysteine proteases, cleaves poly(ADP-ribose) polymerase and sterol regulatory element binding proteins during apoptosis. CPP32 normally exists in the cytosol as a 32-kDa inactive precursor and only becomes activated when cells are undergoing apoptosis. The activation is a proteolytic event that generates a p20/p11 heterodimer. We report here the identification, purification, and characterization of a hamster CPP32-activating protease (CAP) that cleaves and activates CPP32. The biochemical properties of CAP suggest that it is another member of the ICE family of proteases. Purified CAP consists of two prominent polypeptides of 19 and 13 kDa. Protein sequencing revealed that CAP is derived from the hamster homolog of Mch2alpha, a member of the ICE family recently identified based on the sequence conservation among the ICE family members. CAP activity is inhibited by CrmA, a cowpox virus protein that prevents host cell apoptosis. CAP itself is also activated through proteolytic cleavage. These data are consistent with the idea that the activation of the ICE family of proteases during apoptosis proceeds through a cascade of proteolytic events.     

Influence of phosphoric acid etching on the dentin bond durability of universal adhesives

This study aimed to compare the microshear bond strength (MSBS) of three universal adhesives and a three-step conventional adhesive to dentin after 24-hour and one-year storage in water. A new fluoride-releasing universal adhesive (Clearfil Universal Bond Quick: CUQ) and two commercially available adhesives (ScotchBond Universal: SBU and All-Bond Universal: ABU) were evaluated with phosphoric acid etching (PA-etch mode) or without it (self-etch mode). All-Bond 3 (AB3) served as control group. After bonding composite cylinders to dentin discs obtained from caries-free human teeth, the specimens were stored in deionized water at 37?°C for either 24 hours or one year (n?=?14) before MSBS measurement. Two-way ANOVA analysis of the results showed that the adhesives, storage time and their interactions had a significant effect on MSBS (p?<?0.01). In self-etch mode, there was no significant difference among universal adhesives at the baseline. In PA-etch, the CUQ and SBU showed significantly higher MSBS compared with AB3 (p?<?0.05). At baseline, no difference was found between the two modes for each universal adhesive (p?>?0.05). After one year, CUQ in self-etch mode showed a slight increase in nominal MSBS (p?>?0.05) and Weibull characteristic strength, which was significantly higher than SBU and ABU in the corresponding mode. There was no difference among the three universal adhesives in PA-etch mode after one year (p?>?0.05). In conclusion, the durability and reliability of dentin bonding with universal adhesives in different application modes depended on the material; and the self-etch approach showed promising results for the tested fluoride-releasing universal adhesive.