The results of a 60 ps molecular dynamics (MD) simulation of (dG)6.(dC)6 including 10 Na+ counterions and 292 water molecules are presented. All backbone angles and helix parameters for the hexamer are reported in this paper along with trajectory plots of selected angles. Hydrogen bonding between the bases along the helical axis was observed to fluctuate with time, showing the dynamic nature of the base-pairing interaction. These fluctuations gave rise to unusual hydrogen-bonding patterns. Good intrastrand base stacking and no interstrand base stacking were also observed. The hexamer minihelix retains an essentially B-DNA conformation throughout the entire simulation even though some helix parameters and backbone angles do not have strict B-DNA values. The most striking feature obtained from the simulation was a high propeller twist, which resulted in a narrow minor groove for the minihelix. It is proposed that (dG)n.(dC)n sequences are resistant to DNAase I because of this narrow minor groove in dilute aqueous solution.

Base Sequence↗

PubMedDate not supplied

Unilateral cortical spreading depression in the rat: effects on feeding, drinking and other behaviors.

The effects of unilateral cortical spreading depression (CSD) on feeding, drinking and other behaviors were studied in rats. CSD waves were elicited by a microinjection of KC1 solution through an implanted cannula. The animal's behavior and slow potential changes (SPC) accompanying CSD were then simultaneously monitored for 20 min. Intensive face grooming was observed while the CSD wave-front propagated across the sensorimotor cortex. Animals commenced their first feeding or drinking shortly after the invasion of the negative SPC into the frontal cortex of Krieg's area 6 and 10 [14] had reached its maximum height. A two-way switching effect between the first feeding or drinking and quiescence (freezing) and a close relationship between their onsets were observed. The results are discussed specifically in relation to cortico-hypothalamic pathways.

Animals↗

PubMedDate not supplied

Expression of bovine cytochrome P450c17 cDNA in Saccharomyces cerevisiae.

We constructed expression plasmids for bovine adrenal cytochrome P450c17 (P450c17) by inserting the corresponding cDNA between the yeast alcohol dehydrogenase I promoter and terminator of the expression vector pAAH5. Plasmids pA alpha 1 and pA alpha 2 contained the entire coding region for bovine P450c17, whereas pAC alpha 1 included the cDNA coding for chimeric P450c alpha consisting of the amino-terminal 45 amino acid residues of rat P450c and the carboxy-terminal 482 amino acid residues of bovine P450c17. The transformed Saccharomyces cerevisiae AH22/pA alpha 1, AH22/pA alpha 2, and AH22/pAC alpha 1 cells produced about 1 x 10(5), 1 x 10(5), and 2 x 10(4) molecules per cell of the corresponding P450 hemoproteins, respectively. On incubation with the cultures of each of the three strains, progesterone was specifically converted into 17 alpha-hydroxyprogesterone, which was not further converted into androstenedione, indicating that the three strains showed 17 alpha-hydroxylase activity, but almost no C17,20-lyase activity. The microsomal fraction prepared from the AH22/pA alpha 1 cells showed 17 alpha-hydroxylase activity toward progesterone and pregnenolone to higher extents, and exhibited C17,20-lyase activity toward 17 alpha-hydroxypregnenolone to a lesser extent and almost no C17,20-lyase activity toward 17 alpha-hydroxyprogesterone. These results indicated that bovine P450c17 synthesized in S. cerevisiae cells manifests the 17 alpha-hydroxylase activity, but not the C17,20-lyase activity.

Adrenal Glands↗

PubMedDate not supplied

Genetically engineered P450 monooxygenases: construction of bovine P450c17/yeast reductase fused enzymes.

Seven P450/reductase fused enzymes were produced in Saccharomyces cerevisiae by expressing fused cDNAs consisting of bovine cytochrome P450c17 (P450c17) and yeast NADPH-cytochrome P450 reductase (reductase). These fused enzymes differed in the length and amino acid sequence of the hinge region between the P450 and reductase moieties. Expression of the fused constructs under the control of the yeast alcohol dehydrogenase I promoter and terminator of expression vector pAAH5 in S. cerevisiae AH22 cells resulted in the production of about 2-8 X 10(4) molecules per cell of the seven corresponding fused enzymes. Six of the fused enzymes incorporated a protoheme, as confirmed by reduced CO-difference spectra. Recombinant yeast strains producing each of the fused hemoproteins showed P450c17-dependent 17 alpha-hydroxylase activity toward progesterone. The most active fused enzyme, delta N23FE, which lacked the amino-terminal 23 amino acids of the reductase, showed about 10 times higher 17 alpha-hydroxylase activity than bovine P450c17, although the fused enzyme (delta N23FE)' with an amino acid sequence in the hinge region different from delta N23FE was less active than delta N23FE. The fused enzyme delta N0FE, consisting of P450c17 and whole reductase, showed about 1.8 times higher activity than bovine P450c17. No activity was found with delta N84FE lacking the amino-terminal 84 amino acids of the reductase moiety. P450c17-dependent C17,(20)-lyase activity toward 17 alpha-hydroxyprogesterone was detected to lesser extents in the recombinant yeast. Fused bovine P450c17/yeast reductase enzymes show enhanced 17 alpha-hydroxylase activity, and the length and amino acid sequence in the hinge region between the P450c17 and yeast reductase moieties can be important for efficient intramolecular electron transfer in the fused enzymes.

Animals↗