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.     

Ram ribosomes are defective proofreaders

We have studied the kinetics of poly(U) translation by three ribosomal ambiguity (Ram) mutants in an in vitro system with performance characteristics similar to those expressed in vivo. The leucine missense frequency supported by Ram ribosomes with tRNALeu2 increases between six and twelve-fold over that of wild-type ribosomes, while the corresponding increase with tRNALeu4 was between four and eight-fold, depending on the rpsD allele. We have used a steady-state assay for proofreading to identify the kinetic lesion responsible for the Ram phenotype. We were unable to detect any difference between Ram and wild-type ribosomes with respect to the initial kinetics of amino-acyl tRNA selection. All of the increased error rates could be associated with a decreased capacity of these Ram ribosomes to discard non-cognate aminoacyl-tRNA by proof reading.     

Regulation of the genes for E. coli DNA gyrase: Homeostatic control of DNA supercoiling

DNA gyrase is the bacterial enzyme responsible for converting circular DNA to a negatively supercoiled form. We show that the synthesis of DNA gyrase is itself controlled by DNA supercoiling; synthesis is highest when the DNA template is relaxed. The rates of synthesis in vivo of both the A and B subunits of DNA gyase are increased up to 10-fold by treatments that block DNA gyrase activity and decrease the supercoiling of intracellular DNA. Similarly, efficient synthesis of both gyrase subunits in a cell-free S-30 extract depends on keeping the closed circular DNA template in a relaxed conformation. The results suggest that DNA supercoiling in E. coli is controlled by a homeostatic mechanism. Synthesis of the RecA protein and several other proteins is also increased by treatments that relax intracellular DNA.     

Isolation and characterization of a 23 kDa protein essential for photosynthetic oxygen evolution

A 23 kDa protein has recently been demonstrated to participate in photosynthetic oxygen evolution by reconstitution experiments on inside-out thylakoid vesicles (Åkerlund H-E, Jansson C and Andersson B (1982) Biochim Biophys Acta 681:1–10). Here we describe the isolation of the 23 kDa protein from a spinach chloroplast extract using ion-exchange chromatography. The protein was obtained in a yield of 25% and with less than 1% of contaminating proteins. The ability of the protein to stimulate oxygen evolution in inside-out thylakoids was preserved throughout the various fractionation steps. The isolated protein was highly water soluble and appeared as a monomer. Its isoelectric point was at pH=7.3. The amino acid composition showed a high content of polar amino acids, resulting in a polarity index of 49%. The isolated protein lacked metals and other prosthetic groups. Its function as a catalytic or regulating subunit in the oxygen evolving complex is discussed.     

Potentiation of antibody responses by specific IgM: specificity and thymus dependency

Modulation of antibody responses induced by IgM directed against the immunogen was investigated. When IgM directed against ox erythrocytes (ORBC) was given together with trinitrophenyl (TNP)-ORBC, the subsequent antibody response to the carrier, ORBC, as well as the response to the hapten, TNP, was potentiated. In contrast, IgG with carrier specificity inhibited both responses. The hapten-specific potentiation was found in both direct and indirect plaques, and was antigen-dose dependent, i.e., no potentiation was found with the lowest antigen doses. The response to 2,4-dinitrophenyl (DNP)-labeled proteins was potentiated by a monoclonal IgM with specificity for the hapten. The effects were observed both in primary and secondary responses. One strict requirement for IgM potentiation to occur was observed. The determinant against which potentiation was achieved had to be physically linked to the determinant against which the IgM was directed, be it hapten or carrier determinants. Thus, irrelevant IgM-antigen complexes were incapable of potentiating the responses. Similar specificity requirements were found for IgG induced suppression of antibody responses. Experiments with nude mice and their euthymic littermates showed that IgM potentiation of antibody production is T-cell dependent. Furthermore, passive transfer of carrier-primed spleen cells together with antigen challenge suggests that IgM potentiation of secondary antibody responses is dependent on specific carrier-primed immune T cells.     

Outbreak of Giardiasis: Effect of a New Antiflagellate Drug,Tinidazole

We describe here an intensive outbreak of mostly symptomatic (90%) Giardia lamblia infestation in a Swedish student group visiting the U.S.S.R. A new antiflagellate drug, ethyl (2-(2-methyl-5-nitro-1-imidazolyl)ethyl) sulphone, tinidazole, Pfizer, was given in a dosage of 150 mg twice daily for seven days to 10 healthy volunteers and to 24 students infested with G. lamblia. The drug was found to be effective in curing giardiasis and in eradicating G. lamblia from the intestinal tract. All the students with symptomatic giardia infestation became free from gastrointestinal disturbance, usually soon after treatment was started. None of the 24 students had G. lamblia in their stools after tinidazole treatment was discontinued or at follow-up. No side effects of the drug were seen and all of the subjects tolerated it very well.     

The partial purification and characterization of cytosol alcohol dehydrogenase from Astasia

1. The cytosol alcohol dehydrogenase (alcohol-NAD oxidoreductase, EC 1.1.1.1) of Astasia longa was partially purified and characterized from cells grown in the presence of air+CO(2) (95:5) or of O(2)+CO(2) (95:5). 2. Under both these growth conditions, the cells contained a fraction, ADHII, which was characterized by its electrophoretic properties, by a high degree of resistance to heat inactivation, by a sharp pH optimum at 8.2 and by its kinetic properties. The estimated molecular weight of this fraction was approx. 150000, which is similar to that of yeast alcohol dehydrogenase. 3. Cells grown in air+CO(2) (95:5) contain another fraction, ADHI, which can be further separated into two subfractions by polyacrylamide-gel electrophoresis and by DEAE-cellulose chromatography. This was termed fraction ;ADHI-air'. 4. In addition to fraction ADHII, cells grown in the presence of O(2) have a twofold increase in fraction ADHI-air activity as well as two new fractions that could not be demonstrated in air-grown cells. These new fractions which we have called fraction ;ADHI-O(2)', account for about 10% of the total activity. 5. The ADHI fractions (air) and (O(2)) have similar broad pH-activity curves and similar kinetic properties, both having a lower K(m) for ethanol and NAD than fraction ADHII. However, they differ from each other with respect to their activity with various substrates. The estimated molecular weight of these two ADHI fractions and their chromatographic behaviour on hydroxyapatite and on DEAE-cellulose also distinguish them.     

Der cis-trans-Positionseffekt in Heterokaryen morphologischer Mutanten von Podospora anserina

Zusammenfassung Der cis-trans-Positionseffekt in Heterokaryen der nicht gekoppelten morphologischen Mutanten z und oct-1 von Podospora anserina wird durch quantitative Kernunterschiede vorgetäuscht.Im cis-Fall führt die Wuchsverminderung von Hyphen mit überwiegendem Gehalt an Doppelmutantenkernen nach Mikrosektorenbildung im Mycel zu einer Selektion der Hyphen mit überwiegendem Wildkerngehalt. Die Doppelmutantenkerne sind nach 6 cm Wuchsstrecke aus dem Mycel verschwunden. Dieses weist daher Wildphänotyp auf.Im trans-Fall wird eine Kernentmischung durch Anastomosenbildung kompensiert, da die entestehenden Kleinsektoren gleich schnell wachsen. Das Mycel bringt daher die oct-1- und z-Merkmale in einem intermediären Phänotyp zum Ausdruck.

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The cis-trans position effect in heterocaryons of morphological mutants of Podospora anserina

Summary The cis-trans position effect of the two non-linked morphological mutants z and oct-1 in heterocaryons of Podospora anserina is caused by quantitative differences of the two types of nuclei.In the cis configuration, there is a growth diminuition of hyphae containing predominantly nuclei of the double mutant type which leads to a selection of hyphae containing mainly the wildtype nuclei. This is due to the formation of microsectors. After 6 cm of mycelial growth the double mutant nuclei are no longer distinguishable; the mycelium adopts the wild phenotype.In the trans configuration, the microsectors of both types of mutant nuclei exhibit the same growth rate; the separation of nuclei is prevented as a result of the formation of hyphal anastomoses. The mycelium shows then an intermediate phenotype with both mutant characteristics.

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