Inhibition of eukaryotic translation by nucleoside 5'-monophosphate analogues of mRNA 5'-cap: changes in N7 substituent affect analogue activity

Nucleotide cap analogues of 7-methylguanosine 5'-monophosphate (m7GMP) were synthesized in which the 7-methyl moiety was replaced with 7-ethyl (e7), 7-propyl (p7), 7-isopropyl (ip7), 7-butyl (b7), 7-isobutyl (ib7), 7-cyclopentyl (cp7), 7-(carboxymethyl) (cm7), 7-benzyl (bn7), 7-(2-phenylethyl) [7-(2-PhEt)], and 7-(1-phenylethyl) [7-(1-PhEt)]. These derivatives were assayed as competitive inhibitors of capped mRNA translation in reticulocyte lysate. We observed that N7 alkyl and alicyclic substituents larger than ethyl significantly decreased the inhibitory activity of these cap analogues presumably by decreasing their affinity for cap binding proteins, which participate in the initiation of translation. This result defined a maximum size for this class of N7 substituents in the nucleotide binding domain of cap binding proteins. Like m7GMP, the N7-substituted GMP derivatives synthesized in this study were found to be predominantly in the anti conformation as determined by proton NMR analyses. However, bn7GMP and 7-(2-PhEt)GMP, which have aromatic N7 substituents, were more effective than m7GMP as competitive inhibitors of translation. The increased affinity of bn7GMP for cap binding proteins was further examined by synthesis of beta-globin mRNA containing 5'-bn7G, 5'-m7G, or 5'-e7G cap structures. These modified mRNAs were tested as translation templates. Messenger RNA capped with bn7G was observed to increase the translation activity of the template 1.8-fold relative to that of its m7G-capped mRNA counterpart. By contrast, e7G-capped mRNA was 25% less active than m7G-capped mRNA.2+V photo-cross-linking of m7G-capped mRNA to cap binding proteins     

Non-glycosidic iridoids from Cymbaria mongolica

Six non-glycosidic iridoids, i.e. (1R,4S,4aS,7S,7aS)-7-hydroxyl-4-hydroxy- methyl-7-methyl-1-methoxyl-1,4,4a,7a-tetrahydrocyclopenta[e]pyran-3-one (1), (1S,4R,4aS,7S,7aS)-7-hydroxyl-4-hydroxymethyl-7-methyl-1-methoxyl-1,4,4a,7a-tetrahydrocyclopenta[e]pyran-3-one (2), (1R,4R,4aS,7S,7aS)-7-hydroxyl-4-hydroxy-methyl-7-methyl-1-methoxyl-1,4,4a,7a-tetrahydrocyclopenta[e]pyran-3-one (3), (1R, 4R, 4aS, 7aS)-4,7-dihydroxymethyl-1-methoxyl-1,4,4a,7a-tetrahydrocyclopenta-6-ene[e]pyran-3-one (4), (1R, 4R, 4aS, 7aS)-4,7-dihydroxymethyl-1-hydroxyl-1,4,4a, 7a-tetrahydrocyclopenta-6-ene[e]pyran-3-one (5), (1R, 4S, 4aS, 7aS)-4,7-dihydroxy-methyl-1-methoxyl-1,4,4a,7a-tetrahydrocyclopenta-6-ene[e]pyran-3-one (6), as well as five known non-glycosidic iridoids mussaenin A (7), gardendiol (8), isoboonein (9), 4-epi-alyxialactone (10) and rehmaglutin D (11) have been isolated from the Chinese medicinal plant Cymbaria mongolica. Their structures were elucidated by spectroscopic methods. These compounds exhibit significant antitumor and antibacterial activity.     

The chemical composition of the cuticular wax of cranberry

Cuticular wax of cranberry fruit (Vaccinium macrocarpon var. Howes) was analysed by thin layer and gas-liquid chromatography in conjunction with mass spectrometry and infrared spectroscopy. The wax was shown to contain n-paraffins (10·7%), n-aldehydes (14·3%), n-alcohols (6·5%), n-fatty acids (9·7%), sterols (5·0%) and pentacyclic triterpene alcohols (5·8%), acetates (6·1%) and acids (30·7%).     

Apolipoprotein A-I alpha -helices 7 and 8 modulate high density lipoprotein subclass distribution.

Mice have a monodisperse high density lipoprotein (HDL) profile, whereas humans have two major subfractions designated HDL(2) and HDL(3). Human apoA-I transgenic mice exhibit a human-like HDL profile, indicating that the amino acid sequence of apoA-I is a determinant of the HDL profile. Comparison of the primary sequence of mouse and human apoA-I and the previously designated "hinge" domain of apoA-I led us to hypothesize that alpha-helices 7 and 8 (7/8) are determinants of HDL subclass distribution. The following proteins were expressed in Escherichia coli: human apoA-I, T7-hAI; mouse apoA-I, T7-mAI; chimeric human apoA-I containing murine helices 7/8 in place of human helices 7/8, T7-hAI(m7/8); and the reciprocal chimera, T7-mAI(h7/8). The recombinant proteins were examined for their association with human plasma HDL subclasses. The results demonstrated that T7-hAI bound HDL(2) and HDL(3) equally well, whereas T7-mAI bound to HDL(2) preferentially. T7-hAI(m7/8) behaved like T7-mAI, and T7-mAI(h7/8) behaved like T7-hAI. Thus, alpha-helices 7/8 are strong contributors to the pattern of HDL subclass association. Self-association, alpha-helicity, cholesterol efflux, and lecithin-cholesterol acyltransferase activity of the recombinant proteins were also assessed. Human apoA-I self-associates more and activates human lecithin-cholesterol acyltransferase better than mouse apoA-I. These differential characteristics of human and mouse apoA-I are not dependent on helices 7/8.     

A ten-residue domain (Y11-A20) in the NH2-terminus modulates membrane association of annexin A7

Annexin A7 (synexin, annexin VII) is postulated to promote membrane fusion during surfactant secretion in alveolar type II cells and catecholamine secretion in adrenal chromaffin cells. Recently, we demonstrated that the 1-29 residues in the NH(2)-terminus could, possibly by interaction with the COOH-terminus, influence the Ca(2+)-dependent membrane binding, aggregation, and fusion properties of annexin A7 (A7). In this study, we further investigated this 29-residue domain by evaluating several deletion and point mutations for membrane-associated functions of A7. In comparison to A7, the mutants lacking 1-29 residues (A7Delta(1-29)) or 1-21 residues (A7Delta(1-21)), but not those lacking 1-10 residues (A7Delta(1-10)) or 21-29 residues (A7Delta(21-29)), showed diminished membrane binding. Segmental deletion of 10-20 residues (A7Delta(10-20)) also decreased the protein binding to membranes. The Ca(2+)-dependent membrane aggregation of PLV with A7Delta(1-29) was maximally diminished but less so with A7Delta(10-20) or A7Delta(1-21) in comparison to that with A7. However, phospholipid vesicle (PVL) aggregation was unaffected with A7Delta(1-10) or A7Delta(21-29). The Ca(2+)-dependent membrane fusion of PLV was also diminished with A7Delta(10-20) and A7Delta(1-29), but not with A7Delta(1-10). Since the mode of annexin A7 association and function with biological membranes could be different, we also evaluated these proteins for functional changes with isolated lung lamellar bodies. In comparison to A7, the binding to lamellar bodies was diminished for A7Delta(1-29) and A7Delta(1-21) but not for A7Delta(1-10). The Ca(2+)-dependent fusion of isolated lamellar bodies with PLV was also diminished with A7Delta(1-29), but not with A7Delta(10-20) or A7Delta(1-21). Taken together, our studies suggest that the 10-residue domain (Y(11)-A(20)) in the NH(2)-terminus modifies the phospholipid binding and aggregation properties of annexin A7. For binding and fusion of biological membranes, the 10-29-residue domain may be required although the annexin A7 properties are primarily modulated through the Y(11)-A(20) domain.     

Effect of bile acid analogs on 7 alpha-dehydroxylase activity in Eubacterium sp. V.P.I. 12708

7 beta-Methyl-chenodeoxycholic acid (7-MeCDC, 3 alpha, 7 alpha-dihydroxy-7 beta-methyl-5 beta-cholan-24-oic acid), 7 alpha-methyl-ursodeoxycholic acid (7-MeUDC, 3 alpha, 7 beta-dihydroxy-7 alpha-methyl-5 beta-cholan-24-oic acid), 7 xi-methyl-lithocholic acid (7-MeLC, 3 alpha-hydroxy-7 xi-methyl-5 beta-cholan-24-oic acid) and ursodeoxycholylsarcosine (UDCS) were tested as inhibitors of bacterial bile acid 7 alpha-dehydroxylase activity. At a concentration of 50 microM, 7-MeCDC and 7-MeUDC inhibited enzyme activity by 66% and 12%, respectively. 7 alpha-Dehydroxylase activity was not inhibited in the presence of 7-MeLC and UDCS. None of the four bile acid analogs tested inhibited the growth of Eubacterium sp. V.P.I. 12708 at concentrations up to 100 microM.     

Characterization of angiotensin-(1-7) receptor subtype in mesenteric arteries

Mesenteric arteries from male Sprague-Dawley rats were mounted in a pressurized myograph system. Ang-(1-7) concentration-dependent responses were determined in arteries preconstricted with endothelin-1 (10(-7)M). The receptor(s) mediating the Ang-(1-7) evoked dilation were investigated by pretreating the mesenteric arteries with specific antagonists of Ang-(1-7), AT(1) or AT(2) receptors. The effects of Ang-(3-8) and Ang-(3-7) were also determined. Ang-(1-7) caused a concentration-dependent dilation (EC(50): 0.95 nM) that was blocked by the selective Ang-(1-7) receptor antagonist D-[Ala(7)]-Ang-(1-7). Administration of a specific antagonist to the AT(2) receptor (PD123319) had no effect. On the other hand, losartan and CV-11974 attenuated the Ang-(1-7) effect. These results demonstrate that Ang-(1-7) elicits potent dilation of mesenteric resistance vessels mediated by a D-[Ala(7)]-Ang-(1-7) sensitive site that is also sensitive to losartan and CV-11974.     

Evidence for a new angiotensin-(1-7) receptor subtype in the aorta of Sprague-Dawley rats

We have recently described, in the mouse aorta, the vasodilator effect of angiotensin-(1-7) (Ang-(1-7)) was mediated by activation of the Mas Ang-(1-7) receptor and that A-779 and D-Pro7-Ang-(1-7) act as Mas receptor antagonists. In this work we show pharmacological evidence for the existence of a different Ang-(1-7) receptor subtype mediating the vasodilator effect of Ang-(1-7) in the aorta from Sprague-Dawley (SD) rats. Ang-(1-7) induced an endothelium-dependent vasodilator effect in aortic rings from SD rats which was inhibited by removal of the endothelium and by L-NAME (100 microM) but not by indomethacin (10 microM). The Ang-(1-7) receptor antagonist D-Pro7-Ang-(1-7) (0.1 microM) abolished the vasodilator effect of the peptide. However, the other specific Ang-(1-7) receptor antagonist, A-779 in concentrations up to 10 microM, did not affect vasodilation induced by Ang-(1-7). The Ang II AT1 and AT2 receptors antagonists CV11974 (0.01 microM) and PD123319 (1 microM), respectively, the bradykinin B2 receptor antagonist HOE 140 (1 microM) and the inhibitor of ACE captopril (10 microM) did not change the effect of Ang-(1-7). Our results show that in the aorta of SD rats, the vasodilator effect of Ang-(1-7) is dependent on endothelium-derived nitric oxide. This effect is mediated by the activation of Ang-(1-7) receptors sensitive to D-Pro7-Ang-(1-7), but not to A-779, which suggests the existence of a different Ang-(1-7) receptor subtype.     

Potentiation of the hypotensive effect of bradykinin by angiotensin-(1-7)-related peptides.

In this study, we evaluated the bradykinin potentiating activity and ACE inhibitory activity of several Ang-(1-7)-related peptides: Ang-(2-7), Ang-(3-7), Ang-(4-7), Ang-(1-6), Ang-(1-5) and the selective antagonist of Ang-(1-7): D-[Ala7]Ang-(1-7) (A-779). In vivo experiments were performed in freely moving Wistar rats. ACE activity was evaluated by a fluorometric assay in rat plasma using Hip-His-Leu as a substrate. Intravenous injections of Ang-(1-7) (2.2 nmol) transformed the effect of a single dose of bradykinin (1 nmol) into the effect produced by a double dose. A similar bradykinin potentiating activity was demonstrated for Ang-(2-7) and Ang-(3-7). On the other hand, Ang-(1-5), Ang-(1-6), Ang-(4-7) and A-779 did not change the hypotensive effect of bradykinin in doses ranging from 8 up to 25 nmols. The hypotensive effect of bradykinin was increased by intravenous infusion (0.3 ng/min) of Ang-(1-7) > Ang-(2-7) > Ang-(3-7). Conversely, Ang-(1-5), Ang-(1-6), Ang-(4-7) or A-779 did not change the hypotensive effect of bradykinin. ACE inhibition with Ang-(1-7) related peptides occurred in the order: Ang-(2-7) > or = Ang-(3-7) > Ang-(1-7) [>] Ang-(1-5) > Ang-(4-7) > or = Ang-(1-6) > or = A-779. A-779 in concentrations up to 10(-5) M did not change the ACE inhibitory activity of Ang-(1-7). These results suggest that Ang-(1-7), Ang-(2-7) and Ang-(3-7) can modulate bradykinin actions in vivo. More important, our data pointed out that alternative mechanisms besides interaction with ACE are required to explain the bradykinin potentiating activity of Ang-(1-7).     

Cytochrome P450scc-dependent metabolism of 7-dehydrocholesterol in placenta and epidermal keratinocytes

The discovery that 7-dehydrocholesterol (7DHC) is an excellent substrate for cytochrome P450scc (CYP11A1) opens up new possibilities in biochemistry. To elucidate its biological significance we tested ex vivo P450scc-dependent metabolism of 7DHC by tissues expressing high and low levels of P450scc activity, placenta and epidermal keratinocytes, respectively. Incubation of human placenta fragments with 7DHC led to its conversion to 7-dehydropregnenolone (7DHP), which was inhibited by dl-aminoglutethimide, and stimulated by forskolin. Final proof for P450scc involvement was provided in isolated placental mitochondria where production of 7DHP was almost completely inhibited by 22R-hydroxycholesterol. 7DHC was metabolized by placental mitochondria at a faster rate than exogenous cholesterol, under both limiting and saturating conditions of substrate transport, consistent with higher catalytic efficiency (k(cat)/K(m)) with 7DHC as substrate than with cholesterol. Ex vivo experiments showed five 5,7-dienal intermediates with MS spectra of dihydroxy and mono-hydroxy-7DHC and retention time corresponding to 20,22(OH)(2)7DHC and 22(OH)7DHC. The chemical structure of 20,22(OH)(2)7DHC was defined by NMR. 7DHP was further metabolized by either placental fragments or placental microsomes to 7-dehydroprogesterone as defined by UV, MS and NMR, and to an additional product with a 5,7-dienal structure and MS corresponding to hydroxy-7DHP. Furthermore, epidermal keratinocytes transformed either exogenous or endogenous 7DHC to 7DHP. 7DHP inhibited keratinocytes proliferation, while the product of its pholytic transformation, pregcalciferol, lost this capability. In conclusion, tissues expressing P450scc can metabolize 7DHC to biologically active 7DHP with 22(OH)7DHC and 20,22(OH)(2)7DHC serving as intermediates, and with further metabolism to 7-dehydroprogesterone and (OH)7DHP.     

Strong, specific, monodentate G-C base pair recognition by N7-inosine derivatives in the pyrimidine.purine-pyrimidine triple-helical binding motif.

The nucleoside analogs 7-(2'-deoxy-alpha-D-ribofuranosyl)hypoxanthine (alpha7H,1), 7-(2'-deoxy-beta-D-ribofuranosyl)hypoxanthine (beta7H,2) and 7-7-(2'-O-methyl-beta-D- ribofuranosyl)hypoxanthine (beta7HOMe,3) were prepared and incorporated into triplex forming oligodeoxynucleotides, designed to bind to DNA in the parallel (pyrimidine.purine-pyrimidine) motif. By DNase I footprinting techniques and UV-melting curve analysis it was found that, at pH 7. 0, the 15mer oligonucleotides d(TTTTTMeCTXTMeCTMeCTMeCT) (MeC = 5-methyl-deoxycytidine, X =beta7H,beta7HOMe) bind to a DNA target duplex forming a H.G-C base triple with equal to slightly increased (10-fold) stability compared to a control oligodeoxynucleotide in which the hypoxanthine residue is replaced by MeC. Remarkably, triple-helix formation is specific to G-C base pairs and up to 40 microM third strand concentration, no stable triplex exhibiting H.A-T, H.T-A or H.C-G base arrangements could be found (target duplex concentration approximately 0.1 nM). Multiply substituted sequences containing beta7H residues either in an isolated [d(TTTTTbeta7HTbeta7HTbeta7HTbeta7HTbeta7HT)] or in a contiguous [d(TTTbeta7Hbeta7Hbeta7Hbeta7HTTTTbeta7HTTT)] manner still form triplexes with their targets of comparable stability as the control (MeC-containing) sequences at pH 7.0 and high salt or spermine containing buffers. General considerations lead to a structural model in which the recognition of the G-C base pair by hypoxanthine takes place via only one H-bond of the N-H of hypoxanthine to N7 of guanine. This model is supported by a molecular dynamics simulation. A general comparison of the triplex forming properties of oligonucleotides containing beta7H with those containing MeC or N7-2'-deoxyguanosine (N7G) reveals that monodentate recognition in the former case can energetically compete with bidentate recognition in the latter two cases.     

Effect of chronic ethanol feeding on oxysterols in rat liver

It was our hypothesis that, as a consequence of increased oxidative stress, cholesterol-derived hydroperoxides and oxysterols are increased in livers of rats exposed to ethanol. To test this we dosed Wistar rats (approximately 0.1 kg initial body weight) with ethanol chronically (rats fed a nutritionally complete liquid diet containing ethanol as 35% of total calories; sampled liver at approximately 6-7 weeks). We measured concentrations of 7 alpha- and 7 beta-hydroperoxycholest-5-en-3 beta-ol (7 alpha-OOH and 7 beta-OOH) as well as 7 alpha- and 7 beta-hydroxycholesterol (7 alpha-OH and 7 beta-OH), and 3 beta-hydroxycholest-5-en-7-one (also termed 7-ketocholesterol; 7-keto). In response to chronic alcohol feeding, there were significant elevations in the concentrations of 7 alpha-OOH (+169%, P = 0.005) and 7 beta-OOH (+199%, P = 0.011). Increases in the concentrations of hepatic 7-keto (+74%, P = 0.01) and decreases in cholesterol (-43%; P = 0.03) also occurred. In contrast, the concentrations of both 7 alpha-OH and 7 beta-OH were not significant (NS). However, when oxysterols in chronic ethanol-fed rats were expressed relative to cholesterol there were significant increases in 7-keto/cholesterol (P = 0.0006), 7 alpha-OH/cholesterol (P = 0.0018) and 7 beta-OH/cholesterol (P = 0.0047). In conclusion, this is the first report of increased 7 alpha-OOH, 7 beta-OOH, and 7-keto in liver of rats and their elevation in chronic experimental alcoholism represent evidence of increased oxidative stress.     

Oligonucleotide duplex stability controlled by the 7-substituents of 7-deazaguanine bases

Oligonucleotides containing 7-substituted 7-deazaguanine residues (7-methyl, 7-iodo) have been synthesized. The self-complemetary octamer d(I⁷c⁷G-C)₄ containing 7-iodo-7-deaza-2′-deoxyguanosine forms a stabilized duplex compared to the parent oligomer d(G-C)₄ (ΔT_m = +10° C). Also the complex between the oligodeoxynucleotide d(I⁷c⁷G₅-G) and poly(C) is stabilized (ΔT_m = +10°) over that of d(c⁷G₅-G) with poly(C).     

Bioconversion of 7-Aminocephalosporanic Acid by Intact Rhodotorula glutinis Cells

When 7-aminocephalosporanic acid (7-ACA) was used as a single carbon source in the enrichment culture medium for screening 7-ACA-degrading microorganisms, pink yeast colonies appeared frequently, and these were identified as Rhodotorula glutinis. These intact R. glutinis cells converted (i) 7-ACA to deacetyl-7-ACA (7-ADACA) and (ii) monochloroacetyl-7-ACA to monochloroacetyl-7-ADACA at sufficiently high levels to be of commercial interest. Acetylation of 7-ADACA to 7-ACA, the reverse reaction of hydrolysis in an organic medium with methyl acetate as an acetyl donor, was also demonstrated.     

Structure of the 5' terminus of hen oviduct lysozyme messenger ribonucleic acid

Lysozyme mRNA (mRNAlys) was purified from hen oviduct poly(A)-containing RNA by hybridization, labeled with NaB[3H]4 and digested with RNase T1. This revealed the presence of equal amounts of two major oligonucleotides having structures of m7Gppp(Np)7 and m7Gppp(Np)4 plus minor amounts of m7Gppp(Np)2 and m7GpppNp. The total mRNAlys contained the cap structures m7Gpppm6Am, m7GpppGm, m7GpppAm, m7GpppCm, m7GpppA, and m7GpppG, in decreasing order of abundance. The m7Gppp(Np)7 oligonucleotide contained only A-caps and the m7Gppp(Np)4, only G-caps. 32P-labeled 5'-terminal T1-oligonucleotides were prepared, and at least 12 different types were observed, the most abundant being m7Gppp(Np)7 and m7Gppp(Np)4. Their sequences were determined to be m7Gppp(m6)AmNmUCCCG and m7GpppGmNmAG. Taken together with the findings of Grez et al. (Grez, M., Land, H., Giesecke, K., Schutz, G., Jung, A., and Sippel, A. E. (1981) Cell 25, 743-752), these results indicate that in the genomic sequence AGCTTGCAGTCCCGT, 52% of the mRNAlys molecules begin at the underlined A residue and 38% at the underlined G residue.     

Effect of bile acid oxazoline derivatives on microorganisms participating in 7 alpha-hydroxyl epimerization of primary bile acids

We tested bile acid oxazoline derivatives of chenodeoxycholic (CDC-OX), 7-ketolithocholic (7-KLC-OX), ursodeoxycholic (UDC-OX), and deoxycholic (DC-OX) as inhibitors of the 7-epimerization of the primary bile acids cholic acid (CA) and CDC in cultures of four species of bacteria and the human fecal flora. The organisms tested elaborate a 7 alpha- and/or 7 beta-hydroxysteroid dehydrogenase (HSDH); they were Escherichia coli (7 alpha-HSDH), Bacteroides fragilis (7 alpha-HSDH), Clostridium absonum (7 alpha- and 7 beta-HSDH) and Eubacterium aerofaciens (7 beta-HSDH). None of the oxazolines affected 7 alpha-OH oxidation of CA or CDC by E. coli or the growth of the organism. All the oxazolines (except UDC-OX) inhibited the growth of B. fragilis and its 7 alpha-HSDH. In contrast, only DC-OX blocked 7 alpha-OH epimerization of CA by C. absonum. Surprisingly, the other three oxazolines enhanced 7 alpha-OH epimerization of CA, but not that of CDC, which was inhibited (CDC-OX greater than 7-KLC-OX much greater than UDC-OX). Enzymic data suggest that CDC-OX in the presence of CA can induce a greater level of both 7 alpha- and 7 beta-HSDH than CA or CDC-OX alone, CDC-OX being more toxic in the presence of CDC. Formation of urso-bile acid from 7-keto substrates by E. aerofaciens is totally blocked by the oxazolines (except UDC-OX). Similarly, suppression of urso-bile acid formation from primary bile acids by the human fecal flora was evident with DC-OX greater than 7-KLC-OX greater than CDC-OX much greater than UDC-OX, the last being ineffective. The inhibitory activity of the oxazolines on the 7-dehydroxylation of primary bile acids by human fecal flora followed the same order.     

Conversion of 7-dehydrocholesterol to 7-ketocholesterol is catalyzed by human cytochrome P450 7A1 and occurs by direct oxidation without an epoxide intermediate

7-Ketocholesterol is a bioactive sterol, a potent competitive inhibitor of cytochrome P450 7A1, and toxic in liver cells. Multiple origins of this compound have been identified, with cholesterol being the presumed precursor. Although routes for formation of the 7-keto compound from cholesterol have been established, we found that 7-dehydrocholesterol (the immediate precursor of cholesterol) is oxidized by P450 7A1 to 7-ketocholesterol (k(cat)/K(m) = 3 × 10(4) m(-1) s(-1)). P450 7A1 converted lathosterol (Δ(5)-dihydro-7-dehydrocholesterol) to a mixture of the 7-keto and 7α,8α-epoxide products (~1:2 ratio), with the epoxide not rearranging to the ketone. The oxidation of 7-dehydrocholesterol occured with predominant formation of 7-ketocholesterol and with the 7α,8α-epoxide as only a minor product; the synthesized epoxide was stable in the presence of P450 7A1. The mechanism of 7-dehydrocholesterol oxidation to 7-ketocholesterol is proposed to involve a Fe(III)-O-C-C(+) intermediate and a 7,8-hydride shift or an alternative closing to yield the epoxide (Liebler, D. C., and Guengerich, F. P. (1983) Biochemistry 22, 5482-5489). Accordingly, reaction of P450 7A1 with 7-[(2)H(1)]dehydrocholesterol yielded complete migration of deuterium in the product 7-ketocholesterol. The finding that 7-dehydrocholesterol is a precursor of 7-ketocholesterol has relevance to an inborn error of metabolism known as Smith-Lemli-Opitz syndrome (SLOS) caused by defective cholesterol biosynthesis. Mutations within the gene encoding 7-dehydrocholesterol reductase, the last enzyme in the pathway, lead to the accumulation of 7-dehydrocholesterol in tissues and fluids of SLOS patients. Our findings suggest that 7-ketocholesterol levels may also be elevated in SLOS tissue and fluids as a result of P450 7A1 oxidation of 7-dehydrocholesterol.     

一例罕见的复杂易位携带者的染色体绘画研究

本文报道了一例罕见的复杂易位男性携带者,结婚８年,其妻连续７次流产、死胎和出生早夭的畸型儿。用染色体显微切割、ＰＣＲ技术构建的人类７号和８号染色体特异性探针地对其进行了染色体绘画研究,分析确定其核型为：４６,ＸＹ,－７,－８,－９,＋ｄｅｒ（７）、ｔ（７;９）（ｑ２２００;ｐ２４）,＋ｄｅｒ（８）ｉｎｖｉｎｓ（８;７）（ｑ２１００;ｑ３１．２ｑ２２００）,＋ｄｅｒ（９）ｔ（９;７）（ｐ２４;ｑ３１．２）．ｉｓｈｄｅｒ（７）ｔ（７;９）（ｗｃｐ７＋）,ｄｅｒ（８）ｉｎｖｉｎｓ（８;７）（ｗｃｐ７＋,ｗｃｐ８＋）,ｄｅｒ（９）ｔ（９;７）（ｗｃｐ７＋）。染色体绘画技术为研究染色体异常提供了一种有效的分子细胞遗传学技术,本文并对携带者复杂易位的发生机理进行了讨论。     

Novel oxysterols observed in tissues and fluids of AY9944-treated rats: a model for Smith-Lemli-Opitz syndrome

Treatment of Sprague-Dawley rats with AY9944, an inhibitor of 3β-hydroxysterol-Δ(7)-reductase (Dhcr7), leads to elevated levels of 7-dehydrocholesterol (7-DHC) and reduced levels of cholesterol in all biological tissues, mimicking the key biochemical hallmark of Smith-Lemli-Opitz syndrome (SLOS). Fourteen 7-DHC-derived oxysterols previously have been identified as products of free radical oxidation in vitro; one of these oxysterols, 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), was recently identified in Dhcr7-deficient cells and in brain tissues of Dhcr7-null mouse. We report here the isolation and characterization of three novel 7-DHC-derived oxysterols (4α- and 4β-hydroxy-7-DHC and 24-hydroxy-7-DHC) in addition to DHCEO and 7-ketocholesterol (7-kChol) from the brain tissues of AY9944-treated rats. The identities of these five oxysterols were elucidated by HPLC-ultraviolet (UV), HPLC-MS, and 1D- and 2D-NMR. Quantification of 4α- and 4β-hydroxy-7-DHC, DHCEO, and 7-kChol in rat brain, liver, and serum were carried out by HPLC-MS using d(7)-DHCEO as an internal standard. With the exception of 7-kChol, these oxysterols were present only in tissues of AY9944-treated, but not control rats, and 7-kChol levels were markedly (>10-fold) higher in treated versus control rats. These findings are discussed in the context of the potential involvement of 7-DHC-derived oxysterols in the pathogenesis of SLOS.