Cloning and characterization of a novel ß-ketoacyl-ACP reductase from Comamonas testosteroni.

Comamonas testosteroni (C. testosteroni) is a gram negative bacterium which can use steroid as a carbon source and degrade steroid with about 20 special enzymes. Most of the enzymes are inducible enzymes. 3-Oxoacyl-ACP reductase (E.C. 1.1.1.100) alternatively known as ß-ketoacyl-ACP reductase (BKR) is involved in fatty acid syntheses. DNA sequence comparison showed that BKR belongs to the short-chain alcohol dehydrogenase (SDR) family. Our results showed that BKR is necessary for the degradation of steroid hormones in C. testosteroni. The DNA fragment of the BKR gene was cloned into an expressional plasmid pET-15b. BKR protein was expressed with 6× His-tag on the N-terminus and the enzyme was purified with Ni-column. Antibodies against BKR were prepared and a new BKR quantitative ELISA was created in our laboratory. The purified BKR is a 30.6 kDa protein on SDS-PAGE. C. testosteroni was induced by testosterone, estradiol, estriol and cholesterol. The expression of BKR was detected with an ELISA. The result showed that the BKR expression could be induced by cholesterol and estriol but not by testosterone and estradiol. BKR gene knock-out mutant (M-C.T.) was prepared by homologous integration. High performance liquid chromatography (HPLC) was used to detect steroid hormone degradation in C. testosteroni ATCC11996 and BKR gene knock-out mutant. We proved that the M-C.T. eliminated of testosterone degradation. Degradations of cholesterol and estradiol were also decreased. We conclude that the novel BKR in C. testosteroni plays an important role in steroid degradation. This work provides some new information of SDR and steroid degradation in C. testosteroni.

Oestetrol stimulates proliferation and oestrogen receptor expression in breast cancer cell lines: comparison of four oestrogens.

OBJECTIVES: Oestetrol (15-alpha-hydroxyoestriol, E4) is an endogenous oestradiol metabolite mainly produced at high concentrations in the fetal liver. In earlier studies E4 was investigated for its use as marker for pregnancies at risk, especially with vascular problems. Some current investigations suggest that the use of E4 in hormone therapy or contraception may have advantages in terms of breast cancer risk when compared to other oestrogens. METHODS: Proliferation of two oestrogen receptor-positive breast cancer cell lines (ZR 75-1 and HCC 1500) was investigated after incubation with oestrone (E1), oestradiol (E2), oestriol (E3), and oestetrol (E4). Receptor expression of oestrogen receptor-alpha (ERα) and -beta (ERß) was determined by Western-Blot. RESULTS: All four oestrogens elicited a significant proliferative stimulation at concentrations of 10(-10) und 10(-9) M as compared to controls. Oestrone displayed a significantly weaker effect than E2. Oestetrol was significantly less effective than E2 at the lower concentration. Expression of ERα and ERß was significantly upregulated by all oestrogens tested, without differences between the latter. CONCLUSIONS: Our results indicate a slightly lower proliferative effect of E4, but only at low concentrations. However, no difference was found regarding receptor expression. Breast cancer risk associated with use of oestetrol should be tested in clinical trials.

Role of Triton X-100 in chemiluminescent enzyme immunoassays capable of diagnosing genetic disorders.

The use of Triton X surfactants in developing 1,1'-oxalylimidazole chemiluminescent enzyme immunoassays (ODI CEIs) with extended linear response range for the quantification of unconjugated estriol (uE3), alpha-fetoprotein (AFP), and human chorionic gonadotropin (hCG) is reported for the first time. The wider linear dynamic range in ODI CLEIA results from Triton X series (e.g., Triton X-100, -114, -405, -705) acting as an inhibitor in the interaction between Amplex Red (hydrophobic substrate) and horseradish peroxidase (hydrophilic enzyme) to produce resorufin (hydrophobic fluorescent dye). Triton X-100 acts as the appropriate inhibitor in ODI CLEIA. The maximum concentrations of AFP and hCG quantified with sandwich ODI CLEIA in the presence of Triton X-100 were 8 times higher than when analyzed with the same system in the absence of Triton X-100. In addition, the lowest concentration of uE3 determined using competitive ODI CLEIA in the presence of Triton X-100 was 20 times lower than that measured with competitive ODI CLEIA in the absence of Triton X-100. These results indicate that rapid quantification of AFP, uE3, and hCG using cost effective and highly sensitive ODI CLEIAs in the presence of Triton X-100 can be applied as an accurate, precise, and reproducible method to diagnose genetic disorders (e.g., trisomy 18 and trisomy 21) in fetuses.

Combination of elevated maternal serum alpha-fetoprotein (MSAFP) and low estriol is highly predictive of anencephaly.

Increased levels of second trimester maternal serum alpha-fetoprotein (MSAFP) have long been established as a marker for neural tube defects (NTDs). In addition, decreased levels of maternal estriol in the third trimester have been reported in pregnancies with anencephalic fetuses. The purpose of this study was to evaluate whether early second trimester unconjugated serum estriol (uE3) is an independent predictor of NTDs. The study included 57,031 patients who underwent maternal serum screening with MSAFP at 14-22 weeks gestation. Of these, 23,415 also had uE3 measurements. There were 63 cases of NTD, an overall incidence of 1.1 per 1,000. Elevated MSAFP (> or =2.5 MOM) was detected in 1,346 patients, 48 of which had NTDs. Decreased uE3 (< or =0.5) was detected in 1,437 patients, 17 of which had NTDs. The incidence of NTDs was significantly higher in patients with low uE3, compared to patients with normal/high uE3 (1.15% vs. 0.09%, P < 001). Finally, 51 patients had both increased MSAFP and decreased uE3; 16 of these had NTDs, 14 of which were anencephalics. In conclusion, both elevated MSAFP and low maternal serum estriol are predictive of NTD but have a low sensitivity. The combination of abnormally elevated MSAFP and low estriol is highly predictive of NTD in particular anencephaly.

Differential diagnosis and management of very low second trimester maternal serum unconjugated estriol levels, with special emphasis on the diagnosis of X-linked ichthyosis.

Incorporation of maternal serum unconjugated estriol into the calculation of risk may increase the yield of serum screening performed during pregnancy for detection of fetal chromosomal and structural anomalies. The differential diagnosis of very low and undetectable levels of unconjugated estriol in maternal serum is discussed, with special emphasis on the prenatal diagnosis of X-linked ichthyosis. The prenatal detection of these findings dictates skilled genetic counseling, targeted sonographic evaluation and examination of fetal karyotype and fetal cDNA for Xp 22.32 with amniotic fluid levels of cortisol, STS, and ASC.