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Elevated plasma aldosterone levels play a detrimental role in certain forms of congestive heart failure and myocardial fibrosis. We proposed aldosterone synthase (CYP11B2) as a novel target for the treatment of these diseases. In this study, the synthesis and biological evaluation of substituted E- and Z-imidazolylmethylenetetrahydronaphthalenes and E- and Z-imidazolylmethyleneindanes (compounds 1a,b−9a,b) is described. The compounds were prepared by a Wittig-like reaction. They were tested for activity using bovine CYP11B and human CYP11B2 expressed in fission yeast and V79 MZh cells. Selectivity was determined toward human CYP11B1, CYP19, and CYP17. Especially in the case of CYP11B1 (steroid 11β-hydroxylase), selectivity is a crucial issue, since sequence homology between this enzyme and the target enzyme is very high (93%). On the basis of the X-ray structure of human CYP2C9, a protein model of CYP11B2 was developed and docking experiments with the title compounds were performed. The biological results revealed highly potent inhibitors of CYP11B2 (IC50 = 4−93 nM). The Z-isomers usually were more active than the corresponding E-isomers. Different inhibitory profiles could be observed:  rather selective inhibitors of CYP11B1, dual inhibitors of both enzymes, and rather selective inhibitors of CYP11B2. The chloro derivative 8b was found to be a highly potent CYP11B2 inhibitor (IC50 = 4 nM) showing a 5-fold selectivity for CYP11B1 (IC50 = 20 nM). This compound could be an interesting lead for further optimization as a therapeutic agent. It also could be used as well as the CYP11B1 selective compounds as a pharmacological tool.

The paper presents a concept and a first realization of a knowledge based off-line programming system. The design is based on an already developed system which was also applied for relevant production tasks. A characteristic feature of the system is a production task specific, dialog oriented user interface. For a flexible realization of the user interface knowledge based approaches have been applied. A concept for a first realization is presented.

The amino acid in position 54 of adrenodoxin is strongly conserved among ferredoxins, consisting of a threonine or serine. Its role was studied by analyzing mutants T54S and T54A of bovine adrenodoxin. Absorption, circular dichroism, fluorescence, and electron paramagnetic resonance spectra of mutant T54S show that this substitution has no influence on the formation and stability of the ferredoxin. The redox potential of this mutant, however, was lowered by 55 mV as compared with native adrenodoxin, indicating a role for this residue in redox potential modulation. Incorporation of the iron-sulfur cluster was not impaired in the T54A mutant, although structural features of the oxidized protein were considerably changed. The decreased stability of the T54A mutant as compared with the wild type and mutant T54S indicates that a hydrogen bond donor at this position stabilizes the protein. Both mutants have been shown to be functionally active. Replacement of threonine 54 by serine or alanine, however, leads to rearrangements at the recognition sites for its redox partners. This is reflected by decreased Km and Kd values of both mutants for the cytochromes P450, whereas only T54A displayed a decreased Km value in cytochrome c reduction. Substrate conversion was accelerated (2.2- and 2.4-fold for mutants T54A and T54S, respectively) in the CYP11B1-, but not in the CYP11A1-dependent reaction.

It has recently been shown that high molecular weight microtubule-associated proteins (HMWP) in the brain are present in dendrites and are absent from axons (Matus et al., 1981, Proc. Natl. Acad. Sci. U. S. A. 78:3010-3014). In this study we followed the appearance of both HMWP and tubulin in the neonatal rat cerebellum by immunoperoxidase staining, concentrating particularly on comparing Purkinje cell dendrites with adjacent granule cell axons. In the axons both immunohistochemically demonstrable tubulin and structurally distinct microtubules are present at all stages of development. By contrast the Purkinje cell dendrites contain better neither tubulin nor microtubules at early stages of their growth. However, immunoperoxidase staining showed that these developing dendrites are rich in HMWP which are particularly concentrated in the dendritic distal regions. HMWP are also present as patches beneath the surface membrane of the cell body before the emergence of dendrites. Based on this data and the well-documented ability of HMWP to promote microtubule assembly, we propose the hypothesis that during the initial phase of Purkinje neuron differentiation HMWP form part of a specialized cytoskeletal structure which acts as a specifier for the development of dendrites as opposed to axons.

An analysis of the electron transfer kinetics from the reduced [2Fe-2S] center of bovine adrenodoxin and its mutants to the natural electron acceptors, cytochromes P450scc and P45011beta, is the primary focus of this paper. A series of mutant proteins with distinctive structural parameters such as redox potential, microenvironment of the iron-sulfur cluster, electrostatic properties, and conformational stability was used to provide more detailed insight into the contribution of the electronic and conformational states of adrenodoxin to the driving forces of the complex formation of reduced adrenodoxin with cytochromes P450scc and P45011beta and electron transfer. The apparent rate constants of P450scc reduction were generally proportional to the adrenodoxin redox potential under conditions in which the protein-protein interactions were not affected. However, the effect of redox potential differences was shown to be masked by structural and electrostatic effects. In contrast, no correlation of the reduction rates of P45011beta with the redox potential of adrenodoxin mutants was found. Compared with the interaction with P450scc, however, the hydrophobic protein region between the iron-sulfur cluster and the acidic site on the surface of adrenodoxin seems to play an important role for precise complementarity in the tightly associated complex with P45011beta.

Site-directed mutagenesis of a domain (amino acids 299-338) aligning to the I-helix region of P450cam, P450BM3 and P450terp was used to investigate the different regioselectivities displayed in the hydroxylation reactions performed by human aldosterone synthase (P450aldo) and 11beta-hydroxylase (P45011beta). The two enzymes are 93% identical and are essential for the synthesis of mineralocorticoids and glucocorticoids in the human adrenal gland. Single replacement of P450aldo residues for P45011 beta-specific residues at positions 296, 301, 302, 320, and 335 only gave rise to slightly increased 11beta-hydroxylase activities. However, a L301P/A320V double substitution increased 11beta-hydroxylase activity to 60% as compared with that of P45011 beta. Additionally substituting Ala-320 for Val-320 of P45011 beta further enhanced this activity to 85%. The aldosterone synthase activities of the mutant P450aldo proteins were suppressed to a varying degree, with triple replacement mutant L301P/E302D/A320V retaining only 10% and double replacement mutant L301P/A320V retaining only 13% of the P450aldo wild type activity. These results demonstrate a switch in regio- and stereoselectivities of the engineered P450aldo enzyme due to manipulation of residues at three critical positions, and we attribute the determination of these features in P450aldo to the structure of a region analogous to the I-helix in P450cam.

Summary In Asia coronary heart disease mortality is almost 10 fold less frequent than in European countries. These findings attract interest to search for different risk factor patterns. From 1982 to 1985 epidemiologic surveys were carried out in China (n=2047), Japan (n=7580) and Germany (n=6052). Healthy, male subjects, aged 30 to 59 years were enrolled. The prevalence rate of hypertension for the Germans was 20% versus 18% for the Japanese, and 11% for the Chinese. About 69% of Chinese, 55% of Japanese, and 37% of Germans were smokers. About 66% of the Germans were overweight (BMI>25), 17% of the Japanese, and 11% of the Chinese. The highest risk group with cholesterol levels of >300 mg/dl included no Chinese subject, 0.1% of the Japanese, but 5% of the Germans. The lipoprotein profiles among the Japanese and the Chinese collectives typically showed antiatherosclerotic characteristics, whereas most Germans exhibited profiles which support development of atherosclerosis. About 36% of the participants from Germany showed 3 or more risk factors accumulated per person (Japan and China 5%). Multifactorial risk factor reduction for Germany is recommended.

The distributions of tubulin and high molecular weight microtubule-associated proteins (HMWPs) in brain were determined by immunoperoxidase histochemistry with specific antisera. Tubulin was found in microtubules of both neurons and glia and both axons and dendrites. HMWPs were found only in neurons where, in all cases examined, they were associated with dendritic microtubules but not those in axons. Both tubulin and HMWPs were also found in postsynaptic densities. These results indicate that brain microtubules vary in chemical composition. The preferential association of HMWPs with dendritic microtubules suggests that they may play a role in distinguishing between dendritic and axonal export routes from the cell body.

We have isolated cDNA clones for two forms of P-450s, P-450ka-1 and P-450ka-2, from a rabbit kidney cDNA library, using the cDNA for rabbit pulmonary cytochrome P-450p-2, a prostaglandin omega-hydroxylase (Matsubara, S., Yamamoto, S., Sogawa, K., Yokotani, N., Fujii-Kuriyama, Y., Haniu, M., Shively, J.E., Gotoh, O., Kusunose, E., and Kusunose, M. (1987) J. Biol. Chem.262, 13366-13371), as a hybridization probe. The cDNAs for P-450ka-1 and P-450ka-2 encode polypeptides of 510 and 511 amino acids, respectively, with sequence similarity of 85% and 87% to P-450p-2. The two deduced primary structures have 87% identity. RNA blot analysis demonstrated that the mRNAs for P-450ka-1 and P-450ka-2 formed single bands at approximately 3.0- and 2.6-kilobase positions, respectively. The mRNA for P-450ka-1 was expressed only in the liver and kidney and was increased remarkably in these tissues by the administration of clofibrate. In contrast, the mRNA for P-450ka-2 was expressed constitutively in the liver, kidney, and small intestine, but its transcription was enhanced only in the liver by clofibrate treatment. Thus, in spite of their high sequence similarity, these P-450 species have different modes of regulatory expression. Comparison of the nucleotide sequences among P-450ka-1, P-450ka-2, and P-450p-2 shows about 90% overall sequence similarity in any pair of the three sequences. Nucleotide replacements are not evenly distributed, but are rather biased. There is a region of approximately 500 base pairs of exceptionally high homology among the three sequences. These results indicate that the gene conversion event occurred during the evolutionary process of these genes.