Characterization of Met-139 as the photolabeled amino acid residue in the steroid binding site of sex hormone binding globulin using delta 6 derivatives of either testosterone or estradiol as unsubstituted photoaffinity labeling reagents.

Immunopurified human sex hormone binding globulin (SHBG) was photoinactivated and photolabeled by radioinert and radioactive photoaffinity labeling steroids delta 6-testosterone (delta 6-T) and delta 6-estradiol (delta 6-E2). The maximal levels of specific incorporation of these two reagents were 0.50 and 0.33 mol of label/mol of SHBG, respectively. Covalently labeled SHBG fractions were citraconylated, reduced, carboxymethylated, and cleaved by trypsin. Separation of tryptic digests by reverse-phase liquid chromatography gave single radioactive peaks at the same retention times with both steroid reagents. However, the two labeled peptidic fractions could be distinguished by capillary electrophoresis and immunodetection with anti-steroid antibodies, whereas the covalent attachment of radioactivity was confirmed by thin-layer chromatography on silica gel. Edman degradation of the two labeled peptides showed a single sequence His-Pro-Ile-([3H]X)-Arg corresponding to the pentapeptide His-Pro-Ile-Met-Arg 136-140 of SHBG sequence. The coincidence, in both cases, of the absence of an identifiable amino acid residue and of the elution of the most intense peak of radioactivity at the fourth cycle of Edman degradation suggests that the same Met-139 residue was labeled by delta 6-[1,2-3H2]T or by delta 6-[17 alpha-3H]E2. Liquid secondary ion mass spectrometry of the two peptides showed [M+H]+ ions at m/z 939.8 or 923.8, corresponding respectively to the addition of delta 6-T or delta 6-E2 to the pentapeptide. The presence of the steroid molecule in the delta 6-[3H]T-pentapeptide conjugate was confirmed by the difference of 2 mass units with the [M+H]+ peak of the delta 6-[4-14C]T-pentapeptide conjugate.     

Proton and carbon-13 nuclear magnetic resonance spectroscopy of diastereoisomeric 3- and 17 beta-tetrahydropyranyl ether derivatives of estrone and estradiol

Protection of 3- and 17 beta-hydroxyl groups of estrone and estradiol as tetrahydropyranyl ether derivatives led to mixtures of 2'(R)- and 2'(S)-diastereoisomers which were separated by crystallization (3-tetrahydropyranyl ethers), or by thin-layer chromatography (17-tetrahydropyranyl ethers), and characterized by 1H and 13C nuclear magnetic resonance (NMR). Assignments for NMR signals of estradiol 3,17 beta-ditetrahydropyranyl ether were facilitated by comparison with those of its 15 zeta, 16 zeta-dideuterio analog and by 2D 1H-13C heteroshift correlation experiments. Diastereoisomers of 3-tetrahydropyranyl ether derivatives could be identified through the 13C NMR doublet signals of the anomeric C-2' and the aromatic C-4 carbon atoms in CDCl3. Diastereoisomers of 17-tetrahydropyranyl ether derivatives were recognized from characteristic modifications of 1H NMR signals of H-2', H-6', H-1, H-17, and 18-CH3 protons as well as from the 13C NMR doublet signals corresponding to C-2', C-4', C-6', C-12, C-13, C-16, and C-17 carbon atoms. Low-temperature experiments showed a splitting of the C-2', C-6', and C-17 13C NMR signals of each of the two 17-tetrahydropyranyl ether isomers. The downfield signal (equatorial conformer) of the three resulting doublets was more intense for the 17-tetrahydropyranyl ether 2'(S)-isomer, whereas the upfield signal (axial conformer) was more intense for the 2'(R)-isomer.     

Isolation of antibodies to steroid hormones by affinity chromatography on antigen-linked sepharose: An efficient electrophoretic elution procedure

An electrophoretic elution procedure of antibodies retained on affinity columns is described. It afforded a 60% recovery of the binding activity of a high affinity (Ka ～ 10¹⁰ M^?1) antiserum to 5α-dihydrotestosterone retained on antigen-linked Sepharose 4B affinity columns. These purified unbound antibodies, (Ka ～ 10¹⁰ M^?1) when applied again on identical antigen-linked affinity columns, were all retained and totally recovered after a new electrophoretic elution. Comparable results were obtained by elution with 1M NH₄OH.The residual 40% binding activity remaining on the antigen-linked Sepharose gel after electrophoretic elution was totally recovered by elution with an excess of 5α-dihydrotestosterone. It corresponded to antibodies of higher affinity (Ka ～ 10¹¹ M^?1). On the other hand the residual 40% fraction of antibodies resistant to NH₄OH elution was denaturated.     

Synthesis and characterization by 1H and 13C nuclear magnetic resonance spectroscopy of 17 alpha-hexanoic derivatives of 5 alpha-dihydrotestosterone and testosterone.

The synthesis and characterization of 17 alpha-(6'-hexanoic acid) derivatives of 5 alpha-dihydrotestosterone and testosterone, useful as ligands for affinity chromatography purification or as precursors for affinity-labeling of androgen-binding proteins, is described. Alkynylation of 3-ethylenedioxy-, 3 beta-hydroxy-, and 3 beta,5-dihydroxy-5 alpha-androstan-17-one precursors with the potassium derivative of 5-hexyn-1-ol led to the corresponding 17 alpha-(6'-hydroxyhex-1'-ynyl) derivatives, which were hydrogenated over 10% Pt-C catalyst to give 17 alpha-(6'-hydroxyhexyl) derivatives. Chromic acid oxidation of the primary hydroxy group of the 3-ethylenedioxy-17-hexyl intermediate into carboxylic acid followed by acid cleavage of the 3-ketal group gave 17 alpha-(5'-carboxypentyl)-5 alpha-dihydrotestosterone, which was also obtained directly by chromic acid oxidation of the 3 beta-hydroxy intermediate. Chromic acid oxidation of the primary hydroxy group of the 3 beta,5 alpha-dihydroxy precursor resulted in a 5 alpha-hydroxy-3-oxo intermediate, which was dehydrated to give 17 alpha-(5'-carboxypentyl)testosterone. The 17 alpha configuration of these derivatives and of synthetic precursors was established by comparing their molecular rotations and their 1H and 13C nuclear magnetic resonance (NMR) spectra including solvent effects, with data reported for 17 alpha- or 17 beta-substituted steroid analogs as well as with 1H and 13C NMR reference data recorded in this work for 17 alpha-ethynyltestosterone, 17 alpha-ethynyl-19-nortestosterone, 17 alpha-ethyl-19-nortestosterone, 17 alpha-methyltestosterone, and 17 alpha-methyl-5 alpha-dihydrotestosterone.     

Synthesis and stereochemistry of C-3- and C-7-linked (O-carboxymethyl)-oximino- and hemisuccinamido derivatives of 5 alpha-dihydrotestosterone.

The 3-(O-carboxymethyl)oximino derivative of 17β-hydroxy-5α-androstan-3-one (5α-dihydrotestosterone) was prepared. Thin-layer chromatography of the corresponding methyl ester showed the presence of two syn (60%) and anti (40%) geometrical isomers of the oxime chain to the C-4 position, which were characterized by ¹³C nmr. The 3β-hemisuccinami-do-5α-androstan-17β-ol was obtained after selective saponification with potassium carbonate of the 17β-hemisuccinate group of the 3,17-dihemi-succinoylated derivative of the previously described 3β-amino-5α-androstan-17β-ol. This 3β-hemisuccinamide was purified as the corresponding methyl ester-17β-acetate and was regenerated after saponification. The 3,3'-ethylenedioxy-7-oxo-5α-androstan-17β-yl acetate was obtained in quantitative yield by catalytic hydrogenation over 10% palladium-oncharcoal of the Δ⁵-7-oxo precursor in a dioxane-ethanol mixture containing traces of pyridine. The exclusive 5α-configuration of this hydrogenated product was established from nmr data and was confirmed by the synthesis of methyl 3,3'-ethylenedioxy-7-oxo-5β-cholan-24-oate as 5β-H-reference compound. The preceding 5α-H-7-ketone was converted into the 7-(O-carboxymethyl)oximino derivative (syn isomer to the C-6 position, exclusively) which was esterified into the corresponding methyl ester. The selective hydrolysis of the 3-ethyleneketal group was achieved by a short treatment with a formic acid-ether 1:1 (v/v) mixture at 20°C. Saponification of the latter reaction product with ethanolic potassium hydroxide gave the 7-(O-carboxymethyl)oximino-17β-hydroxy-5α-androstan-3-one derivative, which was characterized as the corresponding methyl ester. The reduction of the oxime of the 5α-H-7-ketone with sodium in ethanol or with lithium-aluminium hydride gave respectively the 7β-amine or the 7α-amine as the major product. The 7β- and 7α-configurations were established from nmr spectra of the corresponding 7-acetamido derivatives. The 7β- and 7α-hemisuccinamido derivatives were prepared from the mixture of 7β- and 7α-amines, as described above for 3-derivatives and were isolated after thin-layer chromatography of the methyl esters, followed by saponification of the corresponding 17β-acetates.     

Structural role of the T94I rhodopsin mutation in congenital stationary night blindness

Congenital stationary night blindness (CSNB) is an inherited and non‐progressive retinal dysfunction. Here, we present the crystal structure of CSNB‐causing T94I^2.61 rhodopsin in the active conformation at 2.3 Å resolution. The introduced hydrophobic side chain prolongs the lifetime of the G protein activating metarhodopsin‐II state by establishing a direct van der Waals contact with K296^7.43, the site of retinal attachment. This is in stark contrast to the light‐activated state of the CSNB‐causing G90D^2.57 mutation, where the charged mutation forms a salt bridge with K296^7.43. To find the common denominator between these two functional modifications, we combined our structural data with a kinetic biochemical analysis and molecular dynamics simulations. Our results indicate that both the charged G90D^2.57 and the hydrophobic T94I^2.61 mutation alter the dark state by weakening the interaction between the Schiff base (SB) and its counterion E113^3.28. We propose that this interference with the tight regulation of the dim light photoreceptor rhodopsin increases background noise in the visual system and causes the loss of night vision characteristic for CSNB patients.     

Functional characterization of an anti-estradiol antibody by site-directed mutagenesis and molecular modelling: modulation of binding properties and prominent role of the V(L) domain in estradiol recognition

The high-affinity monoclonal anti-estradiol antibody 9D3 presents a specificity defect towards estradiol-3-sulphate and 3-glucuronide conjugates incompatible with use in direct immunoassays. The corresponding single-chain variable fragment (scFv), cloned and produced in E. coli, exhibited a 10-fold lower affinity for estradiol (K(a)=1.2 x 10(9) M (-1)) and a slightly increased specificity defect for the 3-position. Site-directed mutagenesis revealed critical residues involved in estradiol recognition and produced mutants exhibiting up to a 3-fold increase of the binding affinity for estradiol and up to a 2-fold decrease of the cross-reactivity with estradiol-3-sulphate. A comparative model of the antibody 9D3-estradiol complex was built in which the estradiol D-ring is buried into the binding pocket while the 3-, 6- and 7-positions are solvent exposed, agreeing with the lack of specificity for these three positions. Two potential alternative orientations of the A-ring, one close to CDR H3 and L2 loops, and the other one close to CDR H2 and L3 loops, have been considered for the docking of estradiol, none of which could be unambiguously privileged taking into account data from cross-reactivity measurements, photolabelling and mutagenesis studies. For both orientations, estradiol is stabilized by hydrogen bonding of the 17beta-OH group with TyrL36, His89 and GlnH35 in the first case, or TyrL36, only, in the second case and by van der Waals contacts from TyrL91 with alpha- or beta-face of estradiol, respectively, and from ValH95 and GlyH97 with the opposite face. To elucidate the molecular basis of antibody 9D3 specificity, as compared with that of another anti-estradiol antibody 15H11, single variable domains (V(H) and V(L)) and scFv hybrids have been constructed. The binding activity of V(L)9D3 as well as the specificity of the V(L)9D3/V(H)15H11 hybrid, both similar to antibody 9D3, revealed a prominent role of V(L) in estradiol recognition. These findings establish premises for antibody engineering to reduce cross-reactivity, especially with estradiol-3-conjugates.     

Phylogenetic studies of the genus <Emphasis Type="Italic">Cebus</Emphasis>(Cebidae-Primates) using chromosome painting and G-banding

Background  

Chromosomal painting, using whole chromosome probes from humans and Saguinus oedipus, was used to establish karyotypic divergence among species of the genus Cebus, including C. olivaceus, C. albifrons, C. apella robustus and C. apella paraguayanus. Cytogenetic studies suggested that the species of this genus have conservative karyotypes, with diploid numbers ranging from 2n = 52 to 2n = 54.     

Synthesis and stereochemistry of 7β- and 7α-amino-, acetamido-, hemisuccinamido- and terephthalamido derivatives of testosterone

The reduction of 3-ethylenedioxy-7-oximino-5-androsten-17β-yl acetate and of its 17β-tetrahydropyranyl ether analog with sodium in ethanol, followed by thin-layer chromatography, allowed the isolation of the corresponding 17β-hydroxy- and 17β-tetrahydropyranyioxy-5-en-7β- and 7α-amines which were also characte-rized as 7-acetamides. The acylation of the two epimeric 17β-hydroxy-5-en-7-amines with succinic anhydride followed by selective saponification of the 17β-hemisuccinate group and diazomethane esterification, gave the corresponding 17β-hydroxy-5-en-7β- and 7α-hemisuccinamido methyl esters characterized also as 17β-acetates. On the other hand, the acylation of the two 17β-tetrahydropyranyl-oxy-5-en-7-amines with the acid chloride of terephthalic acid monomethyi ester led to the more rigid 7β- and 7α-terephthalamido methyl ester side-chains. The acidolysis of the 3-ethyleneketal protecting group of the preceding 5-en-7-N-acyl derivatives regenerated the 4-en-3-oxo function while the 17β-tetrahydropyranyl ether group was cleaved simultaneously into the 17β-alcohol. The four desired 7β- and 7α-hemisuccinamido- and terephthalamido carboxylic side-chain derivatives of 17β-hydroxy-4-androsten-3-one (testosterone) were finally obtained by saponification of the corresponding methyl esters.     

Intravenous injection of human sex steroid hormone-binding globulin in mouse decreases blood clearance rate and testicular accumulation of orally administered [2-125I]iodobisphenol A

Bisphenol A, an environmental compound with estrogenic activity, has been shown to bind human sex steroid hormone-binding globulin (hSHBG), the main plasma transport protein which regulates the metabolism of androgens and estrogens and limits their access to target organs. The present study was conducted to determine whether physiologically relevant concentrations of hSHBG can influence the blood clearance rate of bisphenol A and its accumulation in the testes. A radioactive [2-125I]iodobisphenol tracer was synthesized with an association constant (Ka) for binding to hSHBG of 0.14 +/- 0.01 x 10(6) M(-1) at 37 degrees C, a value much lower than for [2-125I]iodoestradiol, which was also synthesized. We used i.v. injection of immunopurified hSHBG in adult male mice to maintain hSHBG levels within the physiologically possible range for humans (27-267 nM) before gavage administration of [2-125I]iodobisphenol or [2-125I]iodoestradiol, for measuring the blood clearance rate of radioactive signal in blood samples taken during the following 120 min. Testicular accumulation of radioactivity was measured 24 h and 48 h after gavage of [2-125]iodobisphenol A. In mice receiving immunopurified hSHBG or vehicle, the time-dependent blood clearance of radioactivity exhibited a bi-exponential decrease which indicated alpha-diffusion and beta-elimination phases for both radioactive ligands. The presence of circulating hSHBG significantly and dose-dependently lowered the clearance rate of radioactivity. However, much higher circulating levels of hSHBG were required to retard the blood clearance of [2-125I]iodobisphenol A as compared to those required for [2-125I]iodoestradiol, in keeping with the important difference in their respective Ka value for binding to SHBG. In addition, mice treated with hSHBG exhibited significantly (P = 0.036) reduced testicular accumulation of radioactivity 24 h and 48 h after ingestion of [2-125I]iodobisphenol A. Provided that the binding properties of bisphenol A for hSHBG are not substantially different from those measured for [2-125I]iodobisphenol A, these findings suggest that, although hSHBG binds 2-mono-iodobisphenol A with a relatively low binding affinity, high enough concentrations of circulating hSHBG (range concentrations between 85 and 267 nM) are potentially able to exert a protective effect against exposure to bisphenol A.