Gene expression profiles of primary retinal pigment epithelial cells from apolipoprotein E knockout and human apolipoprotein E2 transgenic mice.

Age-related macular degeneration (AMD) causes visual impairment in the elderly. In non-neovascular AMD, studies involving human subjects have suggested potential involvement of aberrant lipid metabolism. However, there have been no reports on gene expression patterns in animal models of non-neovascular AMD with abnormal lipid metabolism such as apolipoprotein E knockout and human apolipoprotein E2 transgenic mice. Transcriptome analysis was performed using retinal pigment epithelium cells of apoE knockout and apolipoprotein E2 mice using microarray analysis. C57BL/6, Rxrb, Pparbp, Vldlr, and Edf1, which are primarily related to lipid metabolism, were upregulated, while Tgfbr1 and Pdgfb, which are related to pathologic angiogenesis in AMD, were downregulated in both types of mice. Apolipoprotein E knockout and apolipoprotein E2 mice showed characteristic gene expression patterns in the transcriptome analysis of primary retinal pigment epithelium cells. These results suggest that specific genes associated with lipid metabolism and angiogenesis are involved in the pathogenesis and progression of AMD.

Models for extradiol cleaving catechol dioxygenases: syntheses, structures, and reactivities of iron(II)-monoanionic catecholate complexes.

Crystallographic and spectroscopic studies of extradiol cleaving catechol dioxygenases indicate that the enzyme-substrate complexes have both an iron(II) center and a monoanionic catecholate. Herein we report a series of iron(II)-monoanionic catecholate complexes, [(L)Fe(II)(catH)](X) (1a, L = 6-Me(3)-TPA (tris(6-methyl-2-pyridylmethyl)amine), catH = CatH (1,2-catecholate monoanion); 1b, L = 6-Me(3)-TPA, catH = DBCH (3,5-di-tert-butyl-1,2-catecholate monoanion); 1c, L = 6-Me(2)-bpmcn (N,N'-dimethyl-N,N'-bis(6-methyl-2-pyridylmethyl)-trans-1,2-diaminocyclohexane), catH = CatH; 1d, L = 6-Me(2)-bpmcn, catH = DBCH), that model such enzyme complexes. The crystal structure of [(6-Me(2)-bpmcn)Fe(II)(DBCH)](+) (1d) shows that the DBCH ligand binds to the iron asymmetrically as previously reported for 1b, with two distinct Fe-O bonds of 1.943(1) and 2.344(1) A. Complexes 1 react with O(2) or NO to afford blue-purple iron(III)-catecholate dianion complexes, [(L)Fe(III)(cat)](+) (2). Interestingly, crystallographically characterized 2d, isolated from either reaction, has the N-methyl groups in a syn configuration, in contrast to the anti configuration of the precursor complex, so epimerization of the bound ligand must occur in the course of isolating 2d. This notion is supported by the fact that the UV-vis and EPR properties of in situ generated 2d(anti) differ from those of isolated 2d(syn). While the conversion of 1 to 2 in the presence of O(2) occurs without an obvious intermediate, that in the presence of NO proceeds via a metastable S = (3)/(2) [(L)Fe(catH)(NO)](+) adduct 3, which can only be observed spectroscopically but not isolated. Intermediates 3a and 3b subsequently disproportionate to afford two distinct complexes, [(6-Me(3)-TPA)Fe(III)(cat)](+) (2a and 2b) and [(6-Me(3)-TPA)Fe(NO)(2)](+) (4) in comparable yield, while 3d converts to 2d in 90% yield. Complexes 2b and anti-2d react further with O(2) over a 24 h period and afford a high yield of cleavage products. Product analysis shows that the products mainly derive from intradiol cleavage but with a small extent of extradiol cleavage (89:3% for 2b and 78:12% for anti-2d). The small amounts of the extradiol cleavage products observed may be due to the dissociation of an alpha-methyl substituted pyridyl arm, generating a complex with a tridentate ligand. Surprisingly, syn-2d does not react with O(2) over the course of 4 days. These results suggest that there are a number of factors that influence the mode and rate of cleavage of catechols coordinated to iron centers.

Subunits of neurosteroid sulfatase from bovine brain.

We have purified the neurosteroid sulfatase (NSS) from Triton X-100 solubilized microsomes of bovine brain about 100-fold. The purified enzyme is composed of two catalytic units (MW: 57 kDa) and two regulatory units (MW: 38 kDa), making it an alpha(2)beta(2) heterotetramer, whose apparent molecular weight was 180 kDa by gel filtration in the presence of Triton X-100.

The length of the polyoxyethylene chain in the Triton X detergents modulates the apparent activation of neurosteroid sulfatase in bovine brain.

The effect of the Triton X series on the solubilization and enzyme activity of neurosteroid sulfatase (NSS) in the bovine midbrain was investigated. Triton X-100 and X165 stimulated NSS activity in the bovine midbrain, while Triton X-305 did not. This apparent activation was attributed to the action of the detergents, and not to the latency of the enzyme or the removal of some inhibitory substance from the microsomes. The maximum stimulation was obtained when the length of the polyoxyethylene chain of the detergent was 16.

Distribution and characterization of neurosteroid sulfatase from the bovine brain.

We investigated the regional and subcellular distribution of neurosteroid sulfatase (NSS) in the bovine brain and its enzymatic properties by using dehydroepiandrosterone sulfate (DHEA-S) as a substrate. Bovine NSS was highly concentrated in the region of the midbrain and in the hypothalamus. The enzyme was found to be a microsomal enzyme. The optimal temperature of the enzyme was 50 degrees C, which was slightly lower than that of other steroid sulfatases. The optimal pH of bovine NSS was 7.4 with a second optimum at pH 4.0. The second optimal pH of 4.0 was the most characteristic property of bovine NSS. Employing DHEA-S as the substrate, apparent Km and Vmax values were 113 +/- 21 microM and 4.1 +/- 0.4 nmol/mg protein/h, respectively, whereas Km and Vmax values were found to be 1.6 +/- 0.2 M and 1.9 +/- 0.3 micromol/mg protein/h with p-nitrophenyl sulfate (NP-S) as the substrate. NSS has thus been shown to have a higher affinity for the steroid sulfate than the phenolic compound. When DHEA-S was used as the substrate, pregnenolone sulfate (Preg-S) was a competitive inhibitor with an apparent Ki value of 46 microM, and NP-S was a non-competitive inhibitor (apparent Ki=12 mM).

Delta 5-3 beta-hydroxysteroid acyl transferase activity in the rat brain.

Pregnenolone and dehydroepiandrosterone accumulate in brain as sulfate and fatty acid esters and unconjugated steroids. The steroid fatty acid ester-synthesizing activity was investigated in rat brain microsomes. Endogenous fatty acids in the microsomal fraction were used for the esterification of steroids. The enzyme system had a pH optimum of 4.5 in acetate buffer with [3H]dehydroepiandrosterone as substrate. The apparent Km was 9.2 +/- 3.1 x 10(-5) M and Vmax was 18.6 +/- 3.4 nmol/h/mg protein (mean +/- SEM). The inhibition constants of pregnenolone and testosterone were 123 and 64 microM, respectively. Results were compatible with a competitive type of inhibition. A high level of synthetic activity was found in the brain of 1- to 3-week-old male rats, which rapidly decreased with aging. Saponification of purified [3H]pregnenolone esters yielded pregnenolone and a mixture of palmitate, oleate, linoleate, stearate, and myristate as the predominant fatty acids. Contrasting with the high rates of esterification of several radioactive delta 5-3 beta-hydroxysteroids or 17 beta-hydroxysteroids, no fatty acid esters of either cholesterol, epitestosterone (with a hydroxyl group at position C-17 alpha), or corticosterone (with hydroxyl groups at C-21 and C-11 beta) were formed in the same incubation conditions.

Neurosteroids: biosynthesis, metabolism and function of pregnenolone and dehydroepiandrosterone in the brain.

Pregnenolone (P) and dehydroepiandrosterone (D) accumulate in the brain as unconjugated steroids and their sulfate (S) and fatty acid (L) esters. The microsomal acyl-transferase activity is highest in immature (1-3 weeks old) male rats. The immunocytochemical and biochemical evidence for P biosynthesis by differentiated oligodendrocytes is reviewed. The importance of P synthesis for its brain accumulation is assessed by the intracysternal injection of the inhibitor aminoglutethimide. Primary glial cell cultures convert P to 20-OH-P, PL, progesterone, 5 alpha-pregnane-3,20-dione and 3 alpha-hydroxy-5 alpha-pregnane-20-one (Polone). Astroglial cell cultures also produce these metabolites, whereas neurons from 17-day mouse embryos only form 20-OH-P. P and D are converted to the corresponding 7 alpha-hydroxylated metabolites by a very active P-450 enzyme from rat brain microsomes. Several functions of neurosteroids are documented. P decreases in olfactory bulb of intact male rats exposed to the scent of estrous females. D inhibits the aggressive behavior of castrated male mice towards lactating female intruders. The D analog 3 beta-methyl-androst-5-en-17-one, which cannot be metabolized into sex steroids and is not demonstrably androgenic or estrogenic is at least as efficient as D. Both compounds elicit a marked decrease of PS in rat brain. The Cl- conductance of gamma-aminobutyric (GABAA) receptor is stimulated by GABA agonists, an effect which is enhanced by Polone and antagonized by PS. Thus, P metabolites in brain as well as steroids of extraencephalic sources may be involved physiologically in GABAA receptor function. The neurosteroids accumulated in brain may be precursors of sex steroid hormones and progesterone receptors have been localized in glial cells. P and D do not bind to any known intracellular receptor. A heat stable P binding protein has been found in brain cytosol with distinct ligand specificity. A binding component specific for steroids sulfates, including Polone S, DS and PS, in the order of decreasing affinity is localized in adult rat brain synaptosomal membranes. Its relationship to the GABAA receptor is under current investigation.

Pregnenolone, dehydroepiandrosterone, and their sulfate and fatty acid esters in the rat brain.

The rat brain contains large amounts of pregnenolone (P) and dehydroepiandrosterone (D) arising from local biosynthetic pathways. We have devised a procedure for the measurement of both "neurosteroids" either unconjugated or released from their sulfate (S) or fatty acid (L) esters. The measurements were performed at the acrophase of the circadian variation of neurosteroids, and confirmed the large accumulation of P (25 +/- 8 ng/g, mean +/- SD) and of PS (19 +/- 6 ng/g) and DS (2.1 +/- 0.5 ng/g) in the brain of adult male rats. We found that fatty acid esters constitute the major species of neurosteroids in brain (PL 46 +/- 14, and DL 36 +/- 7 ng/g, in adult males). The levels of P and DS were increased by daily injection of vehicle to intact males, whereas castration, without or with testosterone or estradiol supplementation (2 mg daily for 7 days), did not produce a significant change of neurosteroids concentrations. Measurements of neurosteroids had not been previously reported in cyclic females. The levels of P, PL, and DS were identical in proestrous females and in intact males, whereas PS (26 +/- 6 ng/g) and DL (50 +/- 16 ng/g) were increased in females. Compared to proestrous females, diestrous females had lower levels of PS (19 +/- 6 ng/g), DS (1.7 +/- 0.4 ng/g), and PL (43 +/- 19 ng/g). These differences suggested a modulatory role of ovarian secretions on the metabolism of neurosteroids.

Oxidoreductive and hydroxylating metabolism of progesterone in rat liver epithelial cell lines.

Mechanisms and sequences of reduction and hydroxylation of progesterone into 6-hydroxypregnanolones were studied in proliferating rat liver epithelial cell cultures. These cell lines had an intense metabolic activity and all the metabolites were unconjugated. The formation of 3 alpha,6 alpha- and 3 beta,6 alpha-dihydroxy-5 alpha-pregnan-20-one was observed when the cells were incubated with progesterone, 5 alpha-pregnane-3,20-dione, 3 alpha- or 3 beta-hydroxy-5 alpha-pregnan-20-one and 6 alpha-hydroxy-5 alpha-pregnane-3,20-dione but not with 6 alpha- or 6 beta-hydroxyprogesterone, 5 beta-pregnane-3,20-dione, 3 alpha- or 3 beta-hydroxy-5 beta-pregnan-20-one. These findings indicate that the potential precursors of the 6 alpha-hydroxypregnanolones have a 5 alpha-configuration. The reduction of 5 alpha-pregnane-3,20-dione at C-3 followed by a 6 alpha-hydroxylation can be postulated as the major, if not the only, metabolic pathway. However, the possibility that 6 alpha-hydroxylation may occur prior to reduction of the C-3 oxo group cannot be entirely ruled out. The stereospecificity of reduction at C-5 and hydroxylation at C-6 are discussed and compared with 6-hydroxylated progesterone metabolites found in man and some other mammals during pregnancy and the neonatal period.