Formation of 5 alpha-products as major C19-steroids in estrogen-responsive mouse Leydig cell tumor lines

Homogenates of estrogen-responsive mouse Leydig cell tumors (T 124958-R and T 22137) or 28- and 120-day-old mouse testes were incubated with [3H]progesterone or [14C]4-androstene-3,17-dione in the presence of NADPH, and progesterone metabolism and enzyme activities were estimated. The growth of T 124958-R tumor transplanted in BALB/c mice was markedly stimulated by estrogenization of host mice, but the growth of T 22137 tumor was evidently suppressed by the estrogenization. The major C21-17-OH-steroids and C19-steroids formed from progesterone by both tumors and the testes of immature mice were 5 alpha-steroids, such as 3 alpha,17-dihydroxy-5 alpha-pregnan-20-one, 5 alpha-androstane-3,17-dione, androsterone, 3 beta-hydroxy-5 alpha-androstan-17-one and 5 alpha-androstane-3 alpha,17 beta-diol. In contrast, the major steroids formed by the testes of adult mice were testosterone and 4-androstene-3,17-dione, and no or little 5 alpha-steroids were produced. 5 alpha-Reductase activities in both tumor cells (40-50 nmol/l X 10(8) cells per h) were also found to be approx. 5-6 times higher than that in Leydig cells of adult mouse testes (8 nmol/l X 10(8) Leydig cells per h), though 17-hydroxylase activity was much higher in the Leydig cells of adult testes (730 nmol/l X 10(8) Leydig cells per h) than in both tumor cells (1-7 nmol/l X 10(8) cells per h). Furthermore, the presence of significant amounts of endogenous androsterone and/or 5 alpha-androstane-3 alpha,17 beta-diol was demonstrated in both tumors by radioimmunoassay. The present results demonstrate for the first time that C19-5 alpha-steroids are major C19-steroid products (immature type of testicular androgen production) in Leydig cell tumor lines.     

4-Azasteroidal 5 alpha-reductase inhibitors without affinity for the androgen receptor

In efforts to develop potent 5 alpha-reductase inhibitors without affinity for the androgen receptor, synthetic 3-oxo-5 alpha-steroids were tested for their ability to inhibit 5 alpha-reductase, using [14C]testosterone as the substrate, and for their ability to inhibit the binding of [3H]5 alpha-dihydrotestosterone to the androgen receptor of rat prostate cytosol. 2',3' alpha-Tetrahydrofuran-2'-spiro-17-(5 alpha-androstan-3-one) is not an inhibitor of 5 alpha-reductase and has a high affinity for the androgen receptor; substitution of the -CH2- at the 4-position with N-H resulted in a good inhibitor of 5 alpha-reductase. The 4-N-CH3 derivative is even more active, whereas the N-CH2-CH3 derivative is inactive. These 4-aza derivatives have much lower affinity for the androgen receptor than the parent compound. The 4-N-H derivatives of several 3-oxo-5 alpha-steroids were found to be 20-100% as potent as their corresponding 4-N-CH3 analogs as inhibitors of 5 alpha-reductase, whereas their androgen receptor affinities were at least 40-fold lower than their 4-N-CH3 analogs. Their 5 beta-isomers did not inhibit either 5 alpha-reductase or the androgen receptor binding of [3H]5 alpha-dihydrotestosterone. Two of these 4-N-H steroids, 17 beta-N,N-diethylcarbamoyl-4-aza-5 alpha-androstan-3-one and 17 beta-N, N-diisopropylcarbamoyl-4-aza-5 alpha-androstan-3-one, are potent 5 alpha-reductase inhibitors with Ki values equal to 29.2 +/- 1.7 and 12.6 +/- 0.8 nM, respectively, but have little affinity for the androgen receptor. The inhibition of 5 alpha-reductase by both compounds is competitive with testosterone. When [3H]testosterone was incubated with minced rat prostate in the presence of either of these two 4-azasteroids, the nuclear concentration of 5 alpha-dihydrotestosterone decreased and that of testosterone increased. The total nuclear uptake of testosterone plus 5 alpha-dihydrotestosterone was not significantly affected. These 4-azasteroids should be useful for investigating the importance of 5 alpha-reductase in androgen action in vivo.     

Studies on the regioselectivity of the Baeyer-Villiger reaction of 3-keto steroids: conformational effects determine the migration aptitude

A detailed study of the Baeyer-Villiger reaction of 3-ketosteroids has been performed by using m-chloroperoxybenzoic and trifluoroperoxyacetic acids as oxidants. The process was fully regiospecific for 3-keto-5alpha-steroids with the employ of both peracids, and only partially regioselective for 3-keto-5beta-steroids by using trifluoroperoxyacetic acid. Interestingly, the reaction resulted completely unselective for 3-keto-5beta-steroids by using m-chloroperoxybenzoic acid. Theoretical studies were performed to explain the regiochemistry of this process, which is suggested to be controlled by conformational effects in the transition state of the Criegee rearrangement.     

Synthesis of 6-formyluridine 5'-monophosphate: 6-formyl-UMP

A synthesis of 6-formyluridine 5'-monophosphate (6-formylUMP) in 6 steps starting from uridine is described. This approach should be applicable to the preparation of other O5'-phosphorylated 6-formylUrds such as 6-formylUDP and 6-formylUTP.     

Nse5/6 inhibits the Smc5/6 ATPase and modulates DNA substrate binding

Eukaryotic cells employ three SMC (structural maintenance of chromosomes) complexes to control DNA folding and topology. The Smc5/6 complex plays roles in DNA repair and in preventing the accumulation of deleterious DNA junctions. To elucidate how specific features of Smc5/6 govern these functions, we reconstituted the yeast holo‐complex. We found that the Nse5/6 sub‐complex strongly inhibited the Smc5/6 ATPase by preventing productive ATP binding. This inhibition was relieved by plasmid DNA binding but not by short linear DNA, while opposing effects were observed without Nse5/6. We uncovered two binding sites for Nse5/6 on Smc5/6, based on an Nse5/6 crystal structure and cross‐linking mass spectrometry data. One binding site is located at the Smc5/6 arms and one at the heads, the latter likely exerting inhibitory effects on ATP hydrolysis. Cysteine cross‐linking demonstrated that the interaction with Nse5/6 anchored the ATPase domains in a non‐productive state, which was destabilized by ATP and DNA. Under similar conditions, the Nse4/3/1 module detached from the ATPase. Altogether, we show how DNA substrate selection is modulated by direct inhibition of the Smc5/6 ATPase by Nse5/6.     

SYNTHESIS OF 6-FORMYLURIDINE 5′-MONOPHOSPHATE: 6-FORMYL-UMP

A synthesis of 6-formyluridine 5′-monophosphate (6-formylUMP) in 6 steps starting from uridine is described. This approach should be applicable to the preparation of other O5′-phosphorylated 6-formylUrds such as 6-formylUDP and 6-formylUTP.     

The Nse5-Nse6 dimer mediates DNA repair roles of the Smc5-Smc6 complex

Stabilization and processing of stalled replication forks is critical for cell survival and genomic integrity. We characterize a novel DNA repair heterodimer of Nse5 and Nse6, which are nonessential nuclear proteins critical for chromosome segregation in fission yeast. The Nse5/6 dimer facilitates DNA repair as part of the Smc5-Smc6 holocomplex (Smc5/6), the basic architecture of which we define. Nse5-Nse6 [corrected] (Nse5 and Nse6) [corrected] mutants display a high level of spontaneous DNA damage and mitotic catastrophe in the absence of the master checkpoint regulator Rad3 (hATR). Nse5/6 mutants are required for the response to genotoxic agents that block the progression of replication forks, acting in a pathway that allows the tolerance of irreparable UV lesions. Interestingly, the UV sensitivity of Nse5/6 [corrected] is suppressed by concomitant deletion of the homologous recombination repair factor, Rhp51 (Rad51). Further, the viability of Nse5/6 mutants depends on Mus81 and Rqh1, factors that resolve or prevent the formation of Holliday junctions. Consistently, the UV sensitivity of cells lacking Nse5/6 can be partially suppressed by overexpressing the bacterial resolvase RusA. We propose a role for Nse5/6 mutants in suppressing recombination that results in Holliday junction formation or in Holliday junction resolution.     

3-keto-5 alpha-steroid-delta 4-dehydrogenase from Nocardia corallina: purification and characterization

The inducible 3-keto-5 alpha-steroid-delta 4-dehydrogenase of Nocardia corallina was purified to homogeneity using affinity chromatography on 19-nortestosterone-17-acetoxyaminoethyl Sepharose 4B. SDS-polyacrylamide gel electrophoresis, gel filtration and spectral analysis of flavin suggest that the purified dehydrogenase is a monomeric protein of Mr 60,000 containing one flavin. It has a typical absorption spectrum of flavoprotein with maxima at 457, 375, and 277 nm. The values shifted to 470 and 395 nm on binding of 19-nortestosterone. The enzyme catalyzed the dehydrogenation of 3-keto-5 alpha-steroid at the 4- and 5-position, e.g. the conversion of 5 alpha-androst-1-ene-3,17-dione to 1,4-androstadiene-3,17-dione with the reduction of phenazine methosulfate. The substrate 3-ketosteroid has essentially the 5 alpha-configuration. The enzyme did not reduce potassium ferricyanide but did reduce cytochrome c at a moderate rate, and exhibited only a weak steroid oxidase activity. Stereochemical study demonstrated that the enzyme abstracts the 4 beta, 5 alpha-hydrogens of the substrate as a hydrogen ion through a protein-based reaction and as a hydride ion by transfer to FAD, respectively. The enzyme oxidizes a wide variety of 3-keto-5 alpha-steroids but not 3 beta-hydroxysteroid. The dehydrogenase also catalyzed steroid transhydrogenation between 3-keto-5 alpha-steroid and 3-keto-1,4-diene-steroid. The properties of this enzyme are compared with those of 3-keto-steroid-delta 1-dehydrogenase.     

Functional Deficits in Pak5, Pak6 and Pak5/Pak6 Knockout Mice

The p21-activated kinases are effector proteins for Rho-family GTPases. PAK4, PAK5, and PAK6 are the group II PAKs associated with neurite outgrowth, filopodia formation, and cell survival. Pak4 knockout mice are embryonic lethal, while Pak5, Pak6, and Pak5/Pak6 double knockout mice are viable and fertile. Our previous work found that the double knockout mice exhibit locomotor changes and learning and memory deficits. We also found some differences with Pak5 and Pak6 single knockout mice and the present work further explores the potential differences of the Pak5 knockout and Pak6 knockout mice in comparison with wild type mice. The Pak6 knockout mice were found to weigh significantly more than the other genotypes. The double knockout mice were found to be less active than the other genotypes. The Pak5 knockout mice and the double knockout mice performed worse on the rotorod test. All the knockout genotypes were found to be less aggressive in the resident intruder paradigm. The double knockout mice were, once again, found to perform worse in the active avoidance assay. These results indicate, that although some behavioral differences are seen in the Pak5 and Pak6 single knockout mice, the double knockout mice exhibit the greatest changes in locomotion and learning and memory.     

Nse5/6 is a negative regulator of the ATPase activity of the Smc5/6 complex

The multi-component Smc5/6 complex plays a critical role in the resolution of recombination intermediates formed during mitosis and meiosis, and in the cellular response to replication stress. Using recombinant proteins, we have reconstituted a series of defined Saccharomyces cerevisiae Smc5/6 complexes, visualised them by negative stain electron microscopy, and tested their ability to function as an ATPase. We find that only the six protein ‘holo-complex’ is capable of turning over ATP and that its activity is significantly increased by the addition of double-stranded DNA to reaction mixes. Furthermore, stimulation is wholly dependent on functional ATP-binding pockets in both Smc5 and Smc6. Importantly, we demonstrate that budding yeast Nse5/6 acts as a negative regulator of Smc5/6 ATPase activity, binding to the head-end of the complex to suppress turnover, irrespective of the DNA-bound status of the complex.     

Mechanisms of inactivation of human S-adenosylhomocysteine hydrolase by 5',5',6',6'-tetradehydro-6'-deoxy-6'-halohomoadenosines

In an effort to design more specific and potent inhibitors of S-adenosylhomocysteine (AdoHcy) hydrolase, we investigated the mechanisms by which 5',5',6', 6'-tetradehydro-6'-deoxy-6'-halohomoadenosines (X = Cl, Br, I) inactivated this enzyme. The 6'-chloro (a) and 6'-bromo (b) acetylenic nucleoside analogues produced partial ( approximately 50%) loss of enzyme activity with a concomitant ( approximately 50%) reduction of E-NAD(+) to E-NADH. In addition, Ade and halide ions were released from the inhibitors in amounts suggestive of a process involving enzyme catalysis. AdoHcy hydrolase, which was inactivated with compound a, was shown to contain 2 mol of the inhibitor covalently bound to Lys318 of two subunits of the homotetramer. These data suggest that the enzyme-mediated water addition at the 5' position of compound a or b produces an alpha-halomethyl ketone intermediate, which is then attacked by a proximal nucleophile (i.e., Lys318) to form the enzyme-inhibitor covalent adduct (lethal event); in a parallel pathway (nonlethal event), addition of water at the 6' position produces an acyl halide, which is released into solution and chemically degrades into Ade, halide ion, and sugar-derived products. In contrast, compound c completely inactivated AdoHcy hydrolase by converting 2 equiv of E-NAD(+) to E-NADH and causing the release of 2 equiv of E-NAD(+) into solution. Four moles of the inhibitor was shown to be tightly bound to the tetrameric enzyme. These data suggest that compound c inactivates AdoHcy hydrolase by a mechanism similar to the acetylenic analogue of Ado described previously by Parry et al. [(1991) Biochemistry 30, 9988-9997].     

Differential expression of CYP6A5 and CYP6A5v2 in pyrethroid-resistant house flies, Musca domestica

Two cytochrome P450 alleles, CYP6A5 and CYP6A5v2, were isolated from a pyrethroid-resistant house fly stain, ALHF. The two alleles shared 98% similarity in amino acid sequence. To understand the importance of these two alleles in resistance and examine the expression profile of the two alleles between resistant and susceptible strains, quantitative real-time PCR (qRT-PCR) was performed and compared with the Northern blot analysis. We found that qRT-PCR was an efficient method to characterize the expression profiles between these two sequence-closely-related P450 genes between resistant and susceptible houses flies. One of them, CYP6A5v2, was constitutively overexpressed in ALHF house flies compared with susceptible house fly strains. Moreover, this gene was predominantly expressed in the abdominal tissues of ALHF, in which the primary detoxification organs of insects are located. However, there was no significant difference in the expression of CYP6A5 between ALHF and susceptible house flies. The genetic linkage analysis was conducted to determine the possible link between the constitutively overexpressed CYP6A5v2 and insecticide resistance. CYP6A5v2 was mapped on autosome 5, which is correlated with the linkage of resistance in ALHF. Taken together, the study suggests the importance of CYP6A5v2 in increasing metabolic detoxification of insecticides in ALHF. The distinct expression of CYP6A5 and CYP6A5v2 in resistant and susceptible house flies implies the functional difference of theses two genes in house flies and suggests that they are two recently diverged P450 genes presented in a single organism.     

Studies on the Synthesis of S 5′,6-Methano-5′-Thiourldines

Abstract

Two approaches to the synthesis of the title compounds are described. In the first route, a reactive 5-oxo-6-methylene pyrimidine intermediate that is generated by treating the bis-acetylated or bis-benzoylated nucleosides 10 and 11 with sodium hydroxide undergoes intramolecular attack by the 5′-thiol group to afford the 5-hydroxy cyclonucleoside 12. In the second and higher yielding approach, the S_5′,6-methano linkage is established by an internal allylic displacement reaction that occurs when the 5-bromo-6-methyl nucleoside 24 is treated with base. The conformational properties of S_5′,6-methano-5′-thiouridine (3) and certain long-range spin-spin couplings observed in the NMR spectra of the intermediate nucleosides are discussed.     

AWP1/ZFAND6 functions in Pex5 export by interacting with cys-monoubiquitinated Pex5 and Pex6 AAA ATPase

During biogenesis of the peroxisome, a subcellular organelle, the peroxisomal-targeting signal 1 (PTS1) receptor Pex5 functions as a shuttling receptor for PTS1-containing peroxisomal matrix proteins. However, the precise mechanism of receptor shuttling between peroxisomes and cytosol remains elusive despite the identification of numerous peroxins involved in this process. Herein, a new factor was isolated by a combination of biochemical fractionation and an in vitro Pex5 export assay, and was identified as AWP1/ZFAND6, a ubiquitin-binding NF-κB modulator. In the in vitro Pex5 export assay, recombinant AWP1 stimulated Pex5 export and an anti-AWP1 antibody interfered with Pex5 export. AWP1 interacted with Pex6 AAA ATPase, but not with Pex1-Pex6 complexes. Preferential binding of AWP1 to the cysteine-ubiquitinated form of Pex5 rather than to unmodified Pex5 was mediated by the AWP1 A20 zinc-finger domain. Inhibition of AWP1 by RNA interference had a significant effect on PTS1-protein import into peroxisomes. Furthermore, in AWP1 knock-down cells, Pex5 stability was decreased, similar to fibroblasts from patients defective in Pex1, Pex6 and Pex26, all of which are required for Pex5 export. Taken together, these results identify AWP1 as a novel cofactor of Pex6 involved in the regulation of Pex5 export during peroxisome biogenesis.     

Inflammation Regulates TMPRSS6 Expression via STAT5

TMPRSS6 is a regulated gene, with a crucial role in the regulation of iron homeostasis by inhibiting hepcidin expression. The main regulator of iron homeostasis, the antimicrobial peptide hepcidin, which also has a role in immunity, is directly upregulated by inflammation. In this study, we analyzed whether inflammation is also a modulator of TMPRSS6 expression in vitro and in vivo and we determined the mechanism of this regulation A Human Hepatoma cell line was treated with interleukin-6 and mice were injected with lipopolysaccharide and TMPRSS6 expression and the regulatory mechanism were addressed. In this study, we demonstrate that inflammation downregulates TMPRSS6 expression in vitro and in vivo. The downregulation of Tmprss6 by inflammation in mice is not dependent on the Bmp-Smad pathway but occurs through a decrease in Stat5 phosphorylation. Moreover, Stat5 positively regulates Tmprss6 expression directly by binding to a Stat5 element located on the Tmprss6 promoter. Importantly, our results highlight the functional role of inflammatory modulation of TMPRSS6 expression in the regulation of hepcidin. TMPRSS6 inhibition via decreased STAT5 phosphorylation may be an additional mechanism by which inflammation stimulates hepcidin expression to regulate iron homeostasis and immunity.     

Regioselective synthesis of 1I,1II,5I,5II,6I,6I,6II,6II-2H8-cellobiose

Partially deuteriated 1,5,6,6-(2)H(4)-d-glucose and 1(I),1(II),5(I),5(II),6(I),6(I),6(II),6(II)-(2)H(8)-d-cellobiose were synthesized in high yields and on a large scale from d-glucose. (2)H enrichment at C-5 and C-6 of each glucopyranosyl unit in excess of 85% and 90%, respectively, was realized by (1)H-(2)H exchange in (2)H(2)O containing deuteriated Raney Ni. Nucleophilic addition of LiAlD(4) to 5,6,6-(2)H(3)-2,3,4,6-tetra-O-benzyl-d-gluconolactone led to a 98% (2)H enrichment at C-1. Deuteriated cellobiose is of interest as building block for the synthesis of a model compound of cellulose I.     

Dissecting molecular differences between Wnt coreceptors LRP5 and LRP6

Low-density lipoprotein receptor-related proteins 5 and 6 (LRP5 and LRP6) serve as Wnt co-receptors for the canonical β-catenin pathway. While LRP6 is essential for embryogenesis, both LRP5 and LRP6 play critical roles for skeletal remodeling, osteoporosis pathogenesis and cancer formation, making LRP5 and LRP6 key therapeutic targets for cancer and disease treatment. LRP5 and LRP6 each contain in the cytoplasmic domain five conserved PPPSPxS motifs that are pivotal for signaling and serve collectively as phosphorylation-dependent docking sites for the scaffolding protein Axin. However existing data suggest that LRP6 is more effective than LRP5 in transducing the Wnt signal. To understand the molecular basis that accounts for the different signaling activity of LRP5 and LRP6, we generated a series of chimeric receptors via swapping LRP5 and LRP6 cytoplasmic domains, LRP5C and LRP6C, and studied their Wnt signaling activity using biochemical and functional assays. We demonstrate that LRP6C exhibits strong signaling activity while LRP5C is much less active in cells. Recombinant LRP5C and LRP6C upon in vitro phosphorylation exhibit similar Axin-binding capability, suggesting that LRP5 and LRP6 differ in vivo at a step prior to Axin-binding, likely at receiving phosphorylation. We identified between the two most carboxyl PPPSPxS motifs an intervening "gap4" region that appears to account for much of the difference between LRP5C and LRP6C, and showed that alterations in this region are sufficient to enhance LRP5 PPPSPxS phosphorylation and signaling to levels comparable to LRP6 in cells. In addition we provide evidence that binding of phosphorylated LRP5 or LRP6 to Axin is likely direct and does not require the GSK3 kinase as a bridging intermediate as has been proposed. Our studies therefore uncover a new and important molecular tuning mechanism for differential regulation of LRP5 and LRP6 phosphorylation and signaling activity.     

Nucleophilic Substitution Reactions of 5-Bromo-6-methyluridines

Abstract

The reactions of the 5-bromo-6-methyl-2′,3′-O-isopropylideneuridines 9 and 10 with a number of nucleophiles in hot DMF have been investigated. With acetate ion as the nucleophile, either the 5-acetoxy- (11,12) or the 6-acetoxymethyl- (15) products can be obtained in modest yield depending upon the exact reaction conditions. With nitrogen nucleophiles (aniline or p-methoxybenzylamine) reaction takes place at the 6-methyl carbon, whereas with sulfur nucleophiles (thiophenol, thioacetate) only the 5-substituted products are obtained.     

Physicochemical and biological properties of 6(1),6(3),6(5)-tri-O-alpha-maltosyl-cyclomaltoheptaose (6(1),6(3),6(5)-tri-O-alpha-maltosyl-beta-cyclodextrin)

A unique multibranched cyclomaltooligosaccharide (cyclodextrin, CD) of 6(1),6(3),6(5)-tri-O-alpha-maltosyl-cyclomaltoheptaose [6(1),6(3),6(5)-tri-O-alpha-maltosyl-beta-cyclodextrin, (G(2))(3)-betaCD] was prepared. The physicochemical and biological properties of (G(2))(3)-betaCD were determined together with those of monobranched CDs (6-O-alpha-D-glucopyranosyl-alpha-cyclodextrin (G(1)-alphaCD), 6-O-alpha-D-glucopyranosyl-beta-cyclodextrin (G(1)-betaCD), and 6-O-alpha-maltosyl-beta-cyclodextrin (G(2)-betaCD)). NMR spectra of (G(2))(3)-betaCD were measured using various 2D NMR techniques. The solubility of (G(2))(3)-betaCD in water and MeOH-water solutions was extremely high in comparison with nonbranched betaCD and was about the same as that of the other monobranched betaCDs. The formation of an inclusion complex of (G(2))(3)-betaCD with stereoisomers (estradiol, retinoic acid, quinine, citral, and glycyrrhetinic acid) depends on the cis-trans isomers of guest compounds. The cis isomers of estradiol, retinoic acid, and glycyrrhetinic acid were included more than their trans isomers, while the trans isomers of citral and quinine fit more tightly than their cis isomers. (G(2))(3)-betaCD was the most effective host compound in the cis-trans resolution of glycyrrhetinic acid. Among the branched betaCDs, (G(2))(3)-betaCD exhibited the weakest hemolytic activity in human erythrocytes and showed negligible cytotoxicity in Caco-2 cells up to 200 microM. These results indicate unique characteristics of (G(2))(3)-betaCD in some biological responses of cultured cells.