Oxysterol 7 alpha-hydroxylase activity by cholesterol 7 alpha-hydroxylase (CYP7A)

A 7 alpha-hydroxylation is necessary for conversion of both cholesterol and 27-hydroxycholesterol into bile acids. According to current theories, cholesterol 7 alpha-hydroxylase (CYP7A) is responsible for the former and oxysterol 7 alpha-hydroxylase (CYP7B) for the latter reaction. CYP7A is believed to have a very high substrate specificity whereas CYP7B is active toward oxysterols, dehydroepiandrosterone, and pregnenolone. In the present study, 7 alpha-hydroxylation of various oxysterols in liver and kidney was investigated. Surprisingly, human cholesterol 7 alpha-hydroxylase, CYP7A, expressed as a recombinant in Escherichia coli and COS cells, was active toward 20(S)-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol. This enzyme has previously been thought to be specific for cholesterol and cholestanol. A partially purified and reconstituted cholesterol 7 alpha-hydroxylase enzyme fraction from pig liver showed 7 alpha-hydroxylase activity toward the same oxysterols as metabolized by expressed recombinant human and rat CYP7A. The 7 alpha-hydroxylase activity toward 20(S)-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol in rat liver was significantly increased by treatment with cholestyramine, an inducer of CYP7A. From the present results it may be concluded that CYP7A is able to function as an oxysterol 7 alpha-hydroxylase, in addition to the previously known human oxysterol 7 alpha-hydroxylase, CYP7B. These findings may have implications for oxysterol-mediated regulation of gene expression and for pathways of bile acid biosynthesis. A possible use of 20(S)-hydroxycholesterol as a marker substrate for CYP7A is proposed.     

Amino Acid Substitutions Improve the Immunogenicity of H7N7HA Protein and Protect Mice against Lethal H7N7 Viral Challenge

Avian influenza A H7N7/NL/219/03 virus creates a serious pandemic threat to human health because it can transmit directly from domestic poultry to humans and from human to human. Our previous vaccine study reported that mice when immunized intranasally (i.n) with live Bac-HA were protected from lethal H7N7/NL/219/03 challenge, whereas incomplete protection was obtained when administered subcutaneously (s.c) due to the fact that H7N7 is a poor inducer of neutralizing antibodies. Interestingly, our recent vaccine studies reported that mice when vaccinated subcutaneously with Bac-HA (H7N9) was protected against both H7N9 (A/Sh2/2013) and H7N7 virus challenge. HA1 region of both H7N7 and H7N9 viruses are differ at 15 amino acid positions. Among those, we selected three amino acid positions (T143, T198 and I211) in HA1 region of H7N7. These amino acids are located within or near the receptor binding site. Following the selection, we substituted the amino acid at these three positions with amino acids found on H7N9HA wild-type. In this study, we evaluate the impact of amino acid substitutions in the H7N7 HA-protein on the immunogenicity. We generated six mutant constructs from wild-type influenza H7N7HA cDNA by site directed mutagenesis, and individually expressed mutant HA protein on the surface of baculovirus (Bac-HA_m) and compared their protective efficacy of the vaccines with Bac-H7N7HA wild-type (Bac-HA) by lethal H7N7 viral challenge in a mouse model. We found that mice immunized subcutaneously with Bac-HA_m constructs T143A or T198A-I211V or I211V-T143A serum showed significantly higher hemagglutination inhibition and neutralization titer against H7N7 and H7N9 viruses when compared to Bac-HA vaccinated mice groups. We also observed low level of lung viral titer, negligible weight loss and complete protection against lethal H7N7 viral challenge. Our results indicated that amino acid substitution at position 143 or 211 improve immunogenicity of H7N7HA vaccine against H7N7/NL/219/03 virus.     

Biophysical and antisense properties of oligodeoxynucleotides containing 7-propynyl-, 7-iodo- and 7-cyano-7-deaza-2-amino-2'-deoxyadenosines.

The synthesis of 7-propynyl-, 7-iodo- and 7-cyano-7-deaza-2-amino-2'-deoxyadenosines is described. The nucleosides were synthesized, functionalized into the phosphoramidites and incorporated into oligodeoxynucleotides. Spectroscopic melting experiments against complementary RNA showed increases of 3-4 degreesC per modification for single substitutions and smaller increases per incorporation for multiple substitutions relative to unmodified control sequences. The 7-propyne and 7-iodo nucleosides were incorporated into antisense sequences targeting the 3'-UTR of murine C- raf mRNA. Both nucleosides demonstrated substitution-dependent potency. The sequences with three and four substitutions of the 7-propyne-7-deaza-2-amino-2'-deoxyadenosine exhibited a 2-3-fold increase in potency over unmodifed controls.     

Evolution of the B7 family: co-evolution of B7H6 and NKp30, identification of a new B7 family member, B7H7, and of B7's historical relationship with the MHC

The B7 family of genes is essential in the regulation of the adaptive immune system. Most B7 family members contain both variable (V)- and constant (C)-type domains of the immunoglobulin superfamily (IgSF). Through in silico screening of the Xenopus genome and subsequent phylogenetic analysis, we found novel genes belonging to the B7 family, one of which is the recently discovered B7H6. Humans and rats have a single B7H6 gene; however, many B7H6 genes were detected in a single large cluster in the Xenopus genome. The B7H6 expression patterns also varied in a species-specific manner. Human B7H6 binds to the activating natural killer receptor, NKp30. While the NKp30 gene is single-copy and maps to the MHC in most vertebrates, many Xenopus NKp30 genes were found in a cluster on a separate chromosome that does not harbor the MHC. Indeed, in all species so far analyzed from sharks to mammals, the number of NKp30 and B7H6 genes correlates well, suggestive of receptor-ligand co-evolution. Furthermore, we identified a Xenopus-specific B7 homolog (B7HXen) and revealed its close linkage to B2M, which we have demonstrated previously to have been originally encoded in the MHC. Thus, our study provides further proof that the B7 precursor was included in the proto MHC. Additionally, the comparative analysis revealed a new B7 family member, B7H7, which was previously designated in the literature as an unknown gene, HHLA2.     

Synthesis of 7alpha-hydroxy-dehydroepiandrosterone and 7beta-hydroxy-dehydroepiandrosterone

The fermentation of dehydroepiandrosterone synthesized from the starting material diosgenin using Mucor racemosus produced 7alpha-hydroxy-dehydroepiandrosterone and 7beta-hydroxy-dehydroepiandrosterone. The bioactivity of the microbial metabolites is also discussed. The species M. racemosus was isolated by screening among stains from soil samples collected from various parts of China.     

Duplex Stability of Oligonucleotides Containing 7-Substituted 7-Deaza- and 8-Aza-7-Deazapurine Nucleosides

Abstract

The synthesis of 7-substituted 7-deaza- and 8-aza-7-deazapurine 2′-deoxyribonucleosides, their incorporation into oligonucleotides, and the stability of corresponding duplexes is described.     

Solvent effects on the stability of A7U7p

The thermodynamics of double-helix formation were measured spectrophotometrically for A7U7 in water at 1 M NaCl and for A7U7p in a variety of solvent mixtures and salt. Comparison of the A7U7 results with calorimetric measurements indicates duplex formation involves intermediate states. For A7U7p between 0.06 and 0.55 M Na+, dTm/d(log [Na+]) = 17.4 degrees C, similar to the value of 19.6 degrees C for poly-(A).poly(U) [Krakauer, H., & Sturtevant, J. M. (1968) Biopolymers 6, 491-512]. At 1 M NaCl, the A7U7p duplex is most stable in 100% water. For 10 mol % solutions, the order for A7U7p duplex stability is ethylene glycol greater than glycerol greater than ethanol greater than 2-propanol greater than dimethyl sulfoxide greater than 1-propanol greater than formamide greater than N,N-dimethylformamide greater than urea greater than dioxane. Comparison of changes in stability and thermodynamic parameters with literature results for proteins suggests proteins and A7U7p interact differently with solvent. The results suggest hydrophobic bonding is not a major contributor to the stability of the A7U7p duplex. Comparisons with bulk solvent surface tension suggest the energy of cavity formation is also not a major contributor to duplex stability.     

Karyotype instability with multiple 7/14 and 7/7 rearrangements

Summary Chromosomes were studied in a mentally retarded boy with microcephaly, growth retardation, facial erythema, café-au-lait spots, and IgA deficiency. In the lymphocytes there was a remarkable tendency to exchange parts of the chromosomes Nos. 7 and 14, the translocations almost exclusively taking place in bands 7p13, 7q32 and 14q11. Seven different types of rearrangements between Nos. 7 and 14, and some other chromosomal aberrations were found. No abnormalities could be detected in the bone marrow. The patient somewhat resembles those affected with ataxia-telangiectasia or with Bloom's syndrome, but on clinical and cytogenetic grounds these disorders could be excluded.7/14 Translocations similar to those found in our patient's lymphocytes have been reported to occur very rarely in the lymphocyte cultures of individuals with apparently normal chromosome constitution. A relationship between these phenomena may exist.     

Generation and characterization of B7-H4/B7S1/B7x-deficient mice

Members of the B7 family of cosignaling molecules regulate T-cell proliferation and effector functions by engaging cognate receptors on T cells. In vitro and in vivo blockade experiments indicated that B7-H4 (also known as B7S1 or B7x) inhibits proliferation, cytokine production, and cytotoxicity of T cells. B7-H4 binds to an unknown receptor(s) that is expressed on activated T cells. However, whether B7-H4 plays nonredundant immune regulatory roles in vivo has not been tested. We generated B7-H4-deficient mice to investigate the roles of B7-H4 during various immune reactions. Consistent with its inhibitory function in vitro, B7-H4-deficient mice mounted mildly augmented T-helper 1 (Th1) responses and displayed slightly lowered parasite burdens upon Leishmania major infection compared to the wild-type mice. However, the lack of B7-H4 did not affect hypersensitive inflammatory responses in the airway or skin that are induced by either Th1 or Th2 cells. Likewise, B7-H4-deficient mice developed normal cytotoxic T-lymphocyte reactions against viral infection. Thus, B7-H4 plays a negative regulatory role in vivo but the impact of B7-H4 deficiency is minimal. These results suggest that B7-H4 is one of multiple negative cosignaling molecules that collectively provide a fine-tuning mechanism for T-cell-mediated immune responses.     

7-substituted 8-aza-7-deazapurines and 2,8-diaza-7-deaza-purines: synthesis of nucleosides and oligonucleotides

7-Substituted 8-aza- 7-deazaadenosines 1a-e were synthesized by Sonogashira cross coupling from the corresponding 7-iodo nucleoside in 36-79% yields. Starting from 7-bromo (or 7-iodo)-8-aza-7-deazaadenine, 2a,b were obtained by acid-catalyzed glycosylation followed by deprotection in 53 and 35% yields, repectively. Compounds 2b was applied to cross coupling reaction to give 2c-d in 34-95% yield. Compounds 2a and 4b were further transformed to the phosphoramidites 5 and 6b in 9 and 49% overall yields, which were incorporated into oligonucleotides.     

Oligodeoxynucleotides containing C-7 propyne analogs of 7-deaza-2'-deoxyguanosine and 7-deaza-2'-deoxyadenosine.

The synthesis, hybridization properties and antisense activities of oligodeoxynucleotides (ODNs) containing 7-(1-propynyl)-7-deaza-2'-deoxyguanosine (pdG) and 7-(1-propynyl)-7-deaza-2'-deoxyadenosine (pdA) are described. The suitably protected nucleosides were synthesized and incorporated into ODNs. Thermal denaturation (Tm) of these ODNs hybridized to RNA demonstrates an increased stability relative to 7-unsubstituted deazapurine and unmodified ODN controls. Antisense inhibition by these ODNs was determined in a controlled microinjection assay and the results demonstrate that an ODN containing pdG is approximately 6 times more active than the unmodified ODN. 7-Propyne-7-deaza-2'-deoxyguanosine is a promising lead analog for the development of antisense ODNs with increased potency.     

7-Ketocholesterol

7-Ketocholesterol is a major oxidation product of cholesterol found in human atherosclerotic plaque and is more atherogenic than cholesterol in some animal studies. 7-Ketocholesterol can inhibit cholesterol 7 alpha-hydroxylase, the rate-limiting step in bile acid biosynthesis, as well as strongly inhibiting HMG-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis. It has even been suggested that 7-ketocholesterol is formed enzymically as an endogenous regulator of cholesterol biosynthesis. However, when tested as a pharmacological cholesterol-lowering agent, inhibition of HMG-CoA reductase was rapidly overcome and the 7-ketocholesterol metabolised. In vitro, 7-ketocholesterol has wide-ranging and potent effects, most of which have the potential to contribute to atherosclerosis. For example, 7-ketocholesterol can be cytotoxic and can induce apoptosis in vascular cells. These effects, either individually or more likely, in combination, all implicate 7-ketocholesterol in the initiation and development of atherosclerosis, but further work is needed to establish whether or not its role is a direct causal one.     

Conversion of 7alpha-hydroxycholesterol and 7alpha-hydroxy-beta-sitosterol to 3alpha, 7alpha-dihydroxy- and 3alpha, 7alpha, 12alpha-trihydroxy-5beta-steroids in vitro.

The metabolism of 7alpha-hydroxycholesterol and 7alpha-hydroxy-beta-sitosterol (24alpha-ethyl-5-cholestene-3beta,7alpha-diol) has been compared in rat liver subcellular fractions. 7alpha-Hydroxy-beta-sitosterol was shown to be metabolized in the same manner as 7alpha-hydroxycholesterol. Thus, the following C29 metabolites have been identified: 24alpha-ethyl-7alpha-hydroxy-4-cholesten-3-one, 24alpha-ethyl-7alpha,12alpha-dihydroxy-4-cholesten-3-one, 24alpha-ethyl-7alpha-hydroxy-5beta-cholestan-3-one, 24alpha-ethyl-5beta-cholestane-3alpha,7alpha-diol, 24alpha-ethyl-7alpha,12alpha-dihydrozy-5beta-cholestan-3-one, and 24alpha-ethyl-5beta-cholestane-3alha,7alpha,12alpha-triol. The C29 compounds were generally less efficient substrates. The most pronounced difference was noted for the delta4-3-oxosteroid 5beta-reductase. Thus, 7alpha-hydroxy-4-cholesten-3-one was three to four times as efficiently reduced as the C29 analog. The oxidation of the 3beta,7alpha-dihydroxy-delta5-steroid to the 7alpha-hydroxy-delta4-3-oxosteroid, the 12alpha-hydroxylation of the 7alpha-hydroxy-delta4-3-oxosteroid, and the reduction of the 7alpha-hydroxy-5beta-3-oxosteroid to the 3alpha,7alpha-dihydroxy-5beta-steroid occurred in up to two times better yields for the C27 steroids.     

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).     

Synthesis of 7 alpha- and 7 beta-spermidinylcholesterol, squalamine analogues

Stereoselective synthesis of squalamine dessulfates analogues, 7 alpha and 7 beta-N-[3N-(4-aminobutyl) aminopropyl]aminocholesterol are reported, using 7 alpha and 7 beta-aminocholesterol as a key intermediate. It's the first example in which the position of spermidine is modified at the steroid ring. These molecules showed a comparable antibacteria and fungi activities to squalamine. Then, they have a cytotoxic activity on a human non-small cell bronchopulmonary carcinoma line (NSCLC-N6).     

7-Deazapurine containing DNA: efficiency of c7GdTP, c7AdTP and c7IdTP incorporation during PCR-amplification and protection from endodeoxyribonuclease hydrolysis.

The enzymatic synthesis of 7-deazapurine nucleoside containing DNA (501 bp) is performed by PCR-amplification (Taq polymerase) using a pUC18 plasmid DNA as template and the triphosphates of 7-deaza-2'-deoxyguanosine (c7Gd), -adenosine (c7Ad) and -inosine (c7Id). c7GdTP can fully replace dGTP resulting in a completely modified DNA-fragment of defined size and sequence. The other two 7-deazapurine triphosphates (c7AdTP) and (c7IdTP) require the presence of the parent purine 2'-deoxyribonucleotides. In purine/7-deazapurine nucleotide mixtures Taq polymerase prefers purine over 7-deazapurine nucleotides but accepts c7GdTP much better than c7AdTP or c7IdTP. As incorporation of 7-deazapurine nucleotides represents a modification of the major groove of DNA it can be used to probe DNA/protein interaction. Regioselective phosphodiester hydrolysis of the modified DNA-fragments was studied with 28 endodeoxyribonucleases. c7Gd is able to protect the DNA from the phosphodiester hydrolysis in more than 20 cases, only a few enzymes (Mae III, Rsa I, Hind III, Pvu II or Taq I) do still hydrolyze the modified DNA. c7Ad protects DNA less efficiently, as this DNA could only be modified in part. The absence of N-7 as potential binding position or a geometric distortion of the recognition duplex caused by the 7-deazapurine base can account for protection of hydrolysis.     

Microsequence analysis of DNA-binding proteins 7a, 7b, and 7e from the archaebacterium Sulfolobus acidocaldarius

DNA-binding proteins in eubacteria, such as Escherichia coli NS1 and NS2, are generally small basic molecules. In contrast, the archaebacterium Sulfolobus acidocaldarius contains three groups of DNA-binding proteins which have molecular masses of 7, 8, and 10 kDa. In the first group, five proteins (7a-7e) have been identified, while in the second and third group only two proteins each are present, denoted 8a and 8b and 10a and 10b, respectively. In this paper, we present the primary structures of proteins 7a, 7b, and 7e from the first group. All three proteins contain lysyl residues which are monomethylated to different extents. The modified lysines are found in the NH2-terminal regions of all 7-kDa proteins and in the COOH-terminal part of protein 7e. The sequences of the 7-kDa group are highly similar to each other. All of these macromolecules have been shown to interact specifically with DNA. Protein 7e of the 7-kDa group shows the tightest binding to DNA.