Effect of calmodulin on aldosterone synthesis by a cytochrome P-45011 beta-reconstituted system from bovine adrenocortical mitochondria

Bovine adrenocortical calmodulin was purified and its general properties were examined. The latter were similar to those of bovine brain calmodulin. When added to a cytochrome P-450(11)beta-reconstituted system in the presence of dilauroylphosphatidylcholine, calmodulin decreased the rate of aldosterone production from corticosterone from 0.8 to 0.1 nmol/(min X nmol P-450), while it increased the rate of 18-hydroxycorticosterone production from 1.8 to 4.6 nmol/(min X nmol P-450). This effect of calmodulin on steroid production was maximum at a concentration of 1 microM, when 1 microM cytochrome P-450(11)beta was used. The effect was dependent on the presence of Ca2+, and maximal response was observed at less than 1 microM Ca2+. There was essentially no difference in the effect when bovine brain calmodulin was used. Calmodulin induced a change in the activity of cytochrome P-450(11)beta in the presence of a wide concentration range of corticosterone as a substrate. As for 18-hydroxycorticosterone production, calmodulin increased both the maximal activity and the apparent Km for corticosterone, but it decreased the apparent Km for adrenodoxin. Adrenodoxin at a concentration of less than 20 microM did not fully abolish the effect of calmodulin. A small type I difference spectrum appeared when calmodulin was added to cytochrome P-450(11)beta. The difference spectrum increased significantly in the presence of both Ca2+ and adrenodoxin. These results suggest that calmodulin interacts with cytochrome P-450(11)beta in the presence of adrenodoxin and then modulates the activity of aldosterone synthesis catalyzed by cytochrome P-450(11) beta.     

Flavanol glucosides from rhubarb and Rhaphiolepis umbellata

Investigations of rhubarb and the bark of Rhaphiolepis umbellata led to the isolation of new flavan-3-ol glucosides. Their structures were elucidated on the basis of ¹H and ¹³C NMR analysis hydrolytic studies as (+)-catechin 5-O-β-d-glucopyranoside and (?)-catechin 7-O-β-d-glucopyranoside.     

Effect of a new synthetic free radical scavenger, 2-octadecyl ascorbic acid, on the mortality in mouse endotoxemia

Oxygen-derived free radicals have been implicated as mediators of cellular injury in several model systems. In order to clarify the role of oxygen radicals in endotoxemia, we measured the serial lipid peroxide changes resulting from systemic radical reactions using a newly developed colormetric method. To determine the effect of a free radical scavenger on mortality in endotoxemia, a new synthetic scavenger, 2-Octadecylascorbic acid (CV-3611), which overcome the detrimental properties (circulation half-life and cell penetration) of native SOD, was used in the model of mouse endotoxemia induced by the i.p. administration of E-coli endotoxin (10 mg/kg). Serial LPO (Lipid Peroxide) changes revealed significant elevations from the basal level of 4.52 +/- 0.79 nmol/ml to 10.5 +/- 2.04 nmol/ml at 2h (P less than 0.05), 12.0 +/- 2.44 nmol/ml at 8h (P less than 0.05), 32.8 +/- 12.5 nmol/ml at 12h (P less than 0.05) and 13.6 +/- 2.40 nmol/ml at 24h (P less than 0.05) following i.p. administration of E-coli. The circulation half life of CV-3611 was checked by a reversed-phase HPLC after 10 mg/kg s.c. administration. The level of CV-3611 reached peak levels of 0.54 +/- 0.10 micrograms/ml at 1h and 0.52 +/- 0.20 micrograms/ml at 2h then gradually decreased to the level of 0.04 +/- 0.004 micrograms/ml at 6h and to a non-detectable level at 24h after s.c. administration. Increased survival was seen at 2 days (P less than 0.001) after E-coli endotoxin administration in the CV-3611 treated group compared to the control group. These results suggest that oxygen derived free radicals contribute to mortality in mouse endotoxemia and that antioxidants such as CV-3611 may provide a new therapeutic avenue by improving survival of patients with gram-negative bacterial sepsis.     

Three extra copies of a C4-related gene in H-2 w7 mice are C4/Slp hybrid genes generated by multiple recombinational events

Mice bearing the H-2 _w7 haplotype have five C4-related genes and constitutively express the Slp antigen. To understand the structure and evolution of the five C4-related genes of the C3H.W7 mouse, we have determined nucleotide sequences of the 5 end region of these genes. A C4/Slp hybrid nature was confirmed for three of five C4-related genes as predicted previously by restriction enzyme analysis. The nucleotide sequences of the 5 flanking regions of these three hybrid genes showed close similarity to that of the C4 gene, while the 3 side of the ninth exon of the three hybrid genes showed close similarity to that of the Slp gene. In contrast, the regions between the first exon and the middle of the ninth exon of the three hybrid genes showed a mosaic structure of C4-like and Slp-like sequences. Moreover, the boundaries of the C4-like and Slp-like sequences were quite different among the three hybrid genes. The pattern of nucleotide sequence diversity in this region among the five C4-related sequences could be mainly explained not by point mutations but by gene conversions or unequal crossovers. These results suggest that multiple genetic recombinational events between two homologous sequences played an important role in the generation and diversification of the extra copies of the C4/Slp gene in the H-2 ^w7 mouse.The nucleotide sequence data reported in this paper have been submitted to the DDBJ, EMBL, and GenBank nucleotide sequence databases and have been assigned the accession numbers D90167-71.     

Aldosterone biosynthesis by a reconstituted cytochrome P-45011 beta system

[3H]Corticosterone was incubated with cytochrome P-45011 beta purified to electrophoretic homogeneity from bovine adrenocortical mitochondria, and the reaction products were analyzed by high performance liquid chromatography. The production of aldosterone (21.2 pmol/nmol P-450/min) and 18-hydroxycorticosterone (1.17 nmol/nmol P-450/min) was observed. When lipidic extracts from mitochondria of bovine adrenocortical zona glomerulosa were added to the reaction mixture, the rate of production of aldosterone was increased 28-fold. When [3H]18-hydroxycorticosterone was incubated with cytochrome P-45011 beta, the amount of aldosterone produced was 55.7 pmol/nmol P-450/min in the absence of the lipidic extracts and the enhancing effect of the lipidic extracts was 4-fold.     

A new biologically active phenolic from Cattleya trianaei

A new phenolic, hydroxyeucomic acid, and dopamine were isolated from Cattleya trianaei and their biological activities examined.     

Aberrant splicing caused by the insertion of the B2 sequence into an intron of the complement C4 gene is the basis for low C4 production in H-2k mice.

The serum level of the fourth component of complement (C4) in mice bearing the H-2k haplotype is only 1/10 to 1/20 of that of non-H-2k mice. We have analyzed C4 cDNA clones from B10.BR(H-2k) mouse liver and found aberrant C4 cDNA which contained a 200-base pair (bp) insertion between the exon 13 and exon 14 encoded sequences in addition to the normal C4 cDNA. The 5' 148 bp and the 3' 52 bp of this insert were derived from the B2 sequence, the short interspersed repeats of mouse genome, and the central part of intron 13, respectively. Sequence analysis of intron 13 of the C4k gene showed the presence of a complete copy of a B2 consensus sequence. The structure of aberrant C4 mRNA indicated that the possible 3' splice site in the B2 sequence and the cryptic 5' splice site in intron 13 were used. Both the insertion of the B2 sequence into intron 13 and the presence of aberrant mRNA in the liver were specific to H-2k-bearing mice, suggesting that the aberrant splicing due to the B2 insertion is the basis for low C4 expression in H-2k mice.