25-hydroxylation of C27-steroids and vitamin D3 by a constitutive cytochrome P-450 from rat liver microsomes

A constitutive cytochrome P-450 catalyzing 25-hydroxylation of C27-steroids and vitamin D3 was purified from rat liver microsomes. The enzyme fraction contained 16 nmol of cytochrome P-450/mg of protein and showed only one protein band with a minimum molecular weight of 51,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified cytochrome P-450 catalyzed 25-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha-diol, 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol, and 1 alpha-hydroxyvitamin D3 up to 50 times more efficiently, and 25-hydroxylation of vitamin D3 about 150 times more efficiently than the microsomes. The cytochrome P-450 showed no detectable 25-hydroxylase activity towards vitamin D2 and was inactive in cholesterol 7 alpha-hydroxylation as well as in 12 alpha- and 26-hydroxylations of C27-steroids. It catalyzed hydroxylations of testosterone and demethylation of ethylmorphine at the same rates as, or lower rates than, microsomes. The 25-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol and vitamin D3 with the purified cytochrome P-450 was not stimulated by addition of phospholipid or cytochrome b5 to the reconstituted system. Emulgen inhibited 25-hydroxylase activity towards both substrates. The possibility that 25-hydroxylation of C27-steroids and vitamin D3 is catalyzed by the same species of cytochrome P-450 is discussed.     

25-Hydroxyvitamin D3-24-hydroxylase in rat kidney mitochondria

Assay conditions for the measurement of 25-hydroxyvitamin D3-24-hydroxylase activity in rat kidney mitochondria have been worked out. The product, 24,25-dihydroxyvitamin D3 was quantitated either by high pressure liquid chromatography or by isotope dilution-mass spectrometry. By these procedures, the enzyme activity could be measured with saturating concentration (greater than 2.5 X 10(-6) M) of substrate. Pretreatment of the animals by aminophylline (Kulkowski, J. A., Chow, T., Martinez, J., and Ghazarian, J. G. (1979) Biochem. Biophys. Res. Commun. 90, 50-57) stimulated the 24-hydroxylase activity in vitro at least 2 to 3-fold. The identity of the product was verified by gas chromatography-mass spectrometry. The rates of the reaction varied between 1.5 and 5 pmol/mg of mitochondrial protein.min (at 25 degrees C), and the K'm was determined to be 4.2 X 10(-7) M. Malate, succinate, and isocitrate were all able to support the reaction. Low O2 tension, CO, KCN, and the uncoupler carbonyl cyanide m-chlorophenylhydrazone inhibited the reaction, while the respiratory inhibitor rotenone had no effect. Metyrapone inhibited the reaction with 50% inhibition at a concentration of 2.5 mumol/ml. The enzyme was found to be localized inside the inner mitochondrial membrane. The results indicate that in the rat the renal mitochondrial 25-hydroxyvitamin D3-24-hydroxylase is a cytochrome P-450 and that the reducing equivalents are primarily supplied by NADPH via the energy-dependent transhydrogenase.     

Rapid colorimetric detection of in vitro amplified DNA sequences

A colorimetric assay to detect immobilized amplified nucleic acids has been designed. This approach provides a rapid assay, suitable for clinical diagnosis, to analyze DNA sequences amplified by the polymerase chain reaction. The specific DNA sequences are captured on a solid support by the use of a recombinant fusion protein consisting of the Escherichia coli lac repressor and staphylococcal protein A. The biotin streptavidin system is used to detect the immobilized material. Positive samples can be analyzed by direct solid-phase sequencing. Here, we show that this nonradioactive concept can be used for analysis of Staphylococci and Streptococci and for specific detection of the protozoa Plasmodium falciparum in clinical samples.     

Rat liver foci study on coexposure with 50 Hz magnetic fields and known carcinogens

A study was performed to investigate possible interactions by magnetic fields (MF) with the processes of initiation and promotion of chemically induced preneoplastic lesions in rat liver. Male Sprague-Dawley rats were subjected to a 70% partial hepatectomy followed after 24 h by i.p. injection of diethylnitrosamine (DENA) as a tumour initiator. Starting one week after the DENA-treatment phenobarbital (PB) was given to promote growth of enzymatically altered foci of liver cells. MF was applied immediately after the partial hepatectomy and continued until sacrifice after 12 weeks of PB exposure. Homogenous horizontal AC magnetic fields with a frequency of 50 Hz and flux densities of 0.5 μT or 0.5 mT were used. The rats coexposed with MF and DENA plus PB did not gain weight as much as the rats exposed to the chemical agents only. The MF-exposure also resulted in a slight reduction in size and numbers of the focal lesions. The results suggest an interaction of MF with the processes of chemical carcinogenesis either as a result of stress or depending on effects on the proliferation of preneoplastic cells. © 1993 Wiley-Liss, Inc.     

Lack of effect on the chromosomal non-disjunction in aged female mice after low dose X-irradiation.

Karyotypes were determined in 1064 embryos of aged C57/BL mothers. The virgin female mice were irradiated with 0, 4, 8 or 16 R of X-rays, respectively, and placed with young untreated males 5 days after irradiation. 10.5-days old embryos were recovered from the uterus. Aneuploid embryos classified as alive (heart beats observed at the dissection) were 1 monosomic in the control group (496 embryos) and 2 trisomics in the irradiated group (568 embryos). The number of aneuploid embryos classified as dead was 4 trisomic cases in the control group and 3 trisomics in the irradiated group. The data indicate that trisomic embryos are not uncommon in the mouse but are eliminated in post-implantation death. In contrast to the results of Yamamoto et al. the present data do not demonstrate an increased frequency of chromosome abnormalities in embryos of aged mice X-irradiated before mating as compared to non-irradiated ones.     

Dysgerminoma in a rhesus monkey: morphologic and biological features.

A female Macaca mulatta was observed for 31 months after the initial surgical removal of an ovarian tumor. Solitary metastatic lesions were surgically removed 26 and 28 months after excision of the primary tumor. The animal was killed after 31 months because of additional metastatic lesions. Histological evaluation by light microscopy was not conclusive in determining the origin of neoplasm. Transmission electron microscopy, lymphocyte marker studies, and hormone assays were utilized to confirm the diagnosis of dysgerminoma.     

Ribosome-bound eukaryotic elongation factor 2 protects 5 S rRNA from modification.

The interaction between eukaryotic elongation factor eEF-2 and reconstituted 80 S ribosomes was investigated by analyzing the accessibility of 5 S ribosomal RNA for chemical and enzymatic modification. Ribosomes reconstituted from derived subunits were modified, and the positions of the modified sites were identified by primer extension using a 5 S rRNA-specific probe. All reactive sites were located between nucleotides 38 and 99, and most of them were found in putative single-stranded regions of the 5 S rRNA. Conversion of the ribosomes to the post-translocation type of particles by treatment with the translational inhibitor ricin resulted in the exposure of 3 additional bases for chemical modification, suggesting that the 5 S rRNA was more exposed in this type of ribosome. After binding of eEF-2 in complex with the non-hydrolyzable GTP analogue guanosine 5'-(beta, gamma-methylene)-triphosphate, most of the exposed bases in the 5 S rRNA were protected against both chemical and enzymatic modification.