Different conversion metabolic rates of testosterone are associated to hormone-sensitive status and -response of human prostate cancer cells

The main goal of the present work was to compare the ability of human prostate cancer (PCa) cells to metabolize testosterone (T) in living conditions. To this end we studied three different human PCa cell lines (LNCaP, DU145 and PC3) having different hormone-sensitive status and capability of response to androgens. We used an original approach which allows the evaluation of conversion metabolic rates in growing cells after administration of labeled steroid precursor (presently T), at physiological concentrations (1–10 nM). Analysis of both precursor degradation and formation of several products was carried out using reverse phase-high performance liquid chromatography (RP-HPLC) and “on line” radioactive detection. Comparison of the three human PCa cells revealed that their metabolic aptitude differed in many respects: (i) rates of precursor degradation, (ii) different products' formation, and (iii) extent of conjugate production. In detail, PC3 cells quickly degraded T and exhibited high formation rates of androstenedione (A-4-ene-Ad); both DU145 and LNCaP cells mostly retained high levels of unconverted T, with a limited production of A-4-ene-Ad and its 17-keto derivatives (if any). Either LNCaP or DU145 cells generated a relatively high amount of dihydrotestosterone (DHT). In contrast, neither DHT nor its main metabolites were detected in PC3 cells at both short and longer incubation times. As expected, T degradation and A-4-ene-Ad production were highly correlated (r = 0.97; P < 0.03); similarly, A-4-ene-Ad and DHT formation showed a negative, significant correlation. Negligible production of conjugates was noted in both PC3 and DU145 cells, whilst it was remarkable in LNCaP cells (ranging from 43 to 57%). Overall, our data indicate that human PCa cells degrade T quite differently, favoring alternatively reductive or oxidative patterns of androgen metabolism.     

17α-Ethyl-5β-estrane-3α,17β-diol, a biological marker for the abuse of norethandrolone and ethylestrenol in slaughter cattle

The metabolism of the illegal growth promoter ethylestrenol (EES) was evaluated in bovine liver cells and subcellular fractions of bovine liver preparations. Incubations with bovine microsomal preparations revealed that EES is extensively biotransformed into norethandrolone (NE), another illegal growth promoter. Furthermore, incubations of monolayer cultures of hepatocytes with NE indicated that NE itself is rapidly reduced to 17α-ethyl-5β-estrane-3α,17β-diol (EED). In vivo tests confirmed that, after administration of either EES or NE, EED is excreted as a major metabolite. Therefore, it was concluded that, both in urine and faeces samples, EED can be used as a biological marker for the illegal use of EES and/or NE. Moreover, by monitoring EED in urine or faeces samples, the detection period after NE administration is significantly prolonged. These findings were further confirmed by three cases of norethandrolone abuse in a routine screening program for forbidden growth promoters.     

Molecular modeling indicates that homodimers form the basis for intermediate filament assembly from human and mouse epidermal keratins

Mammalian epidermal keratin molecules adopt rod-shaped conformations that aggregate to form cytoplasmic intermediate filaments. To investigate these keratin conformations and the basis for their patterns of molecular association, graphical methods were developed to relate known amino acid sequences to probable spacial configurations. The results support the predominantly α-helical conformation of keratin chains, interrupted by short non-α-helical linkages. However, it was found that many of the linkages have amino acid sequences typical of β-strand conformations. Space-filling atomic models revealed that the β-strand sequences would permit the formation of 2-chain and 4-chain cylindrical β-helices, fully shielding the hydrophobic amino acid chains that alternate with hydrophilic residues in these sequences. Because of the locations of the β-helical regions in human and mouse stratum corneum keratin chains, only homodimers of the keratins could interact efficiently to form 2-chain and 4-chain β-helices. Tetramers having the directions and degrees of overlap of constituent dimers that have been identified by previous investigators are also predicted from the interactions of β-helical motifs. Heterotetramers formed from dissimilar homodimers could combine, through additional β-helical structures, to form higher oligomers having the dimensions seen in electron microscopic studies. Previous results from chemical crosslinking studies can be interpreted to support the concept of homodimers rather than heterodimers as the basis for keratin filament assembly. © 1995 Wiley-Liss, Inc.     

Reconstitution of the recombinant human γ-tubulin ring complex

Cryo-electron microscopy recently resolved the structure of the vertebrate γ-tubulin ring complex (γ-TuRC) purified from Xenopus laevis egg extract and human cells to near-atomic resolution. These studies clarified the arrangement and stoichiometry of γ-TuRC components and revealed that one molecule of actin and the small protein MZT1 are embedded into the complex. Based on this structural census of γ-TuRC core components, we developed a recombinant expression system for the reconstitution and purification of human γ-TuRC from insect cells. The recombinant γ-TuRC recapitulates the structure of purified native γ-TuRC and has similar functional properties in terms of microtubule nucleation and minus end capping. This recombinant system is a central step towards deciphering the activation mechanisms of the γ-TuRC and the function of individual γ-TuRC core components.     

Peptides containing the sulfonamide junction: Synthesis,structure, and conformation of Z-Tau-Pro-Phe-NHiPr

The taurine (Tau) containing tripeptide derivative Z-Tau-Pro-Phe-NHiPr (1) has been synthesized as suitable sulfonamido-pseudopeptide model to investigate formation and conformational properties of folded secondary structures stabilized by intramolecular H bonds directly involving the sulfonamide junction. In the crystal the pseudopeptide 1 adopts a type I β-turn with the Pro and Phe residues located at the (i + 1) and (i + 2) corner positions, respectively. The turn is stabilized by a 4 → 1 H bond engaging one of the SO₂ oxygen atoms and the isopropylamide NH. In CDCl₃ solution the β-turn folding is accompanied by a γ-turn centered at the Pro and involving a 3 → 1 H bond between the SO₂ and the Phe NH. A comparison of the structural and conformational properties found in 1 with those of the already known sulfonamido-pseudopeptides, with particular reference to the models containing the Tau-Pro junction, is also reported. © 1997 John Wiley & Sons, Inc. Biopoly 41: 555–567, 1997.     

A novel approach to predicting protein structural classes in a (20–1)-D amino acid composition space

The development of prediction methods based on statistical theory generally consists of two parts: one is focused on the exploration of new algorithms, and the other on the improvement of a training database. The current study is devoted to improving the prediction of protein structural classes from both of the two aspects. To explore a new algorithm, a method has been developed that makes allowance for taking into account the coupling effect among different amino acid components of a protein by a covariance matrix. To improve the training database, the selection of proteins is carried out so that they have (1) as many non-homologous structures as possible, and (2) a good quality of structure. Thus, 129 representative proteins are selected. They are classified into 30 α, 30 β, 30 α + β, 30 α/β, and 9 ζ (irregular) proteins according to a new criterion that better reflects the feature of the structural classes concerned. The average accuracy of prediction by the current method for the 4 × 30 regular proteins is 99.2%, and that for 64 independent testing proteins not included in the training database is 95.3%. To further validate its efficiency, a jackknife analysis has been performed for the current method as well as the previous ones, and the results are also much in favor of the current method. To complete the mathematical basis, a theorem is presented and proved in Appendix A that is instructive for understanding the novel method at a deeper level. © 1995 Wiley-Liss, Inc.     

From methyl -glucopyranoside to methyl -allopyranoside via the Mitsunobu reaction

Methyl β- -glucopyranoside reacted with a 4-molar excess of the Mitsunobu reagents (triphenylphosphine–diethyl azodicarboxylate–benzoic acid) under Weinges et al. [Carbohydr. Res., 164 (1987) 453–458] conditions to furnish four differently benzoylated methyl β- -allopyranosides in a very good overall yield. The same reaction applied to methyl α- -glucopyranoside yielded allosides in a low yield and nine other sugar products. These results give an insight into the course of the Mitsunobu esterification–inversion reaction.     

Coexistence of β1- and β2-Adrenoceptors in the Melanophore of the Goby Tridentiger obscurus*

The effects of β-adrenergic agonists and antagonists on the pigmentary state of denervated melanophores in isolated, split, caudal fins of the goby Tridentiger obscurus were examined to investigate the function and the subtype of the β-adrenoceptors of the melanophores. Salbutamol, terbutaline, and dobutamine partially inhibited the pigment-aggregating response of melanophores to norepinephrine. The effects of these β-agonists were inhibited by propranolol. It was confirmed that the melanophores possess both α-and β-adrenoceptors, and that the activation of the β-adrenoceptors induces the dispersion of pigment in the melanophores. Norepinephrine, epinephrine, isoproterenol, dobutamine, salbutamol, and terbutaline evoked the dispersion of pigment in the melanophores in which pigment had previously been aggregated by treatment with verapamil in the presence of phentolamine. The pigment-dispersing effects of two β₁-selective agonists, norepinephrine and dobutamine, were effectively inhibited by metoprolol, a selective antagonist of β₁-receptors. By contrast, the pigment-dispersing effects of two β₂-selective agonists, salbutamol and terbutaline, were not inhibited by metoprolol. Both the effects of nonselective agonists, epinephrine and isoproterenol, were partially inhibited by metoprolol. The actions of all of the β-agonists used were effectively inhibited by propranolol, and they were partially inhibited by butoxamine. These results suggest coexistence of β₁- and β₂-adrenoceptors in the melanophores. The relative numbers of β₁- and β₂-adrenoreceptors as a percentage of the total population of β-adrenoceptors were estimated to be 18.6% and 81.4%, respectively, from analyses of Hofstee plots of the effects of the β-agonists on the melanophores in the presence of butoxamine or metoprolol.     

Cloning of a neoteleost (Oreochromis mossambicus) pro-opiomelanocortin (POMC) cDNA reveals a deletion of the γ-melanotropin region and most of the joining peptide region: implications for POMC processing

A signature feature of tetrapod pro-opiomelanocortin (POMC) is the presence of three melantropin (MSH) coding regions (α-MSH, β-MSH, γ-MSH). The MSH duplication events occurred early during the radiation of the jawed vertebrates well over 400 million years ago. However, in at least one order of modern bony fish (subdivision Teleostei; order Salmoniformes; i.e. salmon and trout) the γ-MSH sequence has been deleted from POMC. To determine whether the γ-MSH deletion has occurred in other teleost orders, a POMC cDNA was cloned from the pituitary of the neoteleost Oreochromis mossambicus (order Perciformes). In O. mossambicus POMC, the deletion is more extensive and includes the γ-MSH sequence and most of the joining peptide region. Because the salmoniform and perciform teleosts do not share a direct common ancestor, the γ-MSH deletion event must have occurred early in the evolution of the neoteleost fishes. The post-translational processing of O. mossambicus POMC occurs despite the fact that the proteolytic recognition sequence, (R/K)-X_n-(R/K) where n can be 0, 2, 4, or 6, a common feature in mammalian neuropeptide and polypeptide hormone precursors, is not present at several cleavage sites in O. mossambicus POMC. These observations would indicate that either the prohormone convertases in teleost fish use distinct recognition sequences or vertebrate prohormone convertases are capable of recognizing a greater number of primary sequence motifs around proteolytic cleavage sites.     

15β-hydroxysteroids (Part IV). Steroids of the human perinatal period: The synthesis of 3α,15β,17α-trihydroxy-5α-pregnan-20-one and its A/B-ring configurational isomers

In recent years several 15β-hydroxysteroids have emerged pathognomonic of adrenal disorders in human neonates of which 3α,15β,17α-trihydroxy-5β-pregnan-20-one (2) was the first to be identified in the urine of newborn infants affected with congenital adrenal hyperplasia. In this investigation we report the synthesis of the three remaining 3ξ,5ξ-isomers, namely 3α,15β,17α-trihydroxy-5α-pregnan-20-one (3), 3β,15β,17α-trihydroxy-5α-pregnan-20-one (7) and 3β,15β,17α-trihydroxy-5β-pregnan-20-one (8) for their definitive identification in pathological conditions in human neonates. 3β,15β-Diacetoxy-17α-hydroxy-5-pregnen-20-one (11), a product of chemical synthesis was converted to the isomeric 3 and 7, while conversion of 15β,17α-dihydroxy-4-pregnen-3,20-dione (4), a product of microbiological transformation, resulted in the preparation of 8. In brief, selective acetate hydrolysis of 11 gave 15β-acetoxy-3β,17α-dihydroxy-5-pregnen-20-one (12) which on catalytic hydrogenation gave 15β-acetoxy-3β,17α-dihydroxy-5α-pregnan-20-one (13) a common intermediate for the synthesis of the 3β(and α),5α-isomers. Hydrolysis of the 15β-acetate gave 7, whereas oxidation with pyridinium chlorochromate gave 15β-acetoxy-17α-hydroxy-5α-pregnan-3,20-dione (14) which on reduction with -Selectride and hydrolysis of the 15β-acetate gave 3. Finally, hydrogenation of 4 gave 15β,17α-dihydroxy-5β-pregnan-3,20-dione (10) which on reduction with -Selectride gave 8.     

Folding type-specific secondary structure propensities of amino acids,derived from α-helical, β-sheet, α/β, and α+β proteins of known structures

Folding type-specific secondary structure propensities of 20 naturally occurring amino acids have been derived from α-helical, β-sheet, α/β, and α+β proteins of known structures. These data show that each residue type of amino acids has intrinsic propensities in different regions of secondary structures for different folding types of proteins. Each of the folding types shows markedly different rank ordering, indicating folding type-specific effects on the secondary structure propensities of amino acids. Rigorous statistical tests have been made to validate the folding type-specific effects. It should be noted that α and β proteins have relatively small α-helices and β-strands forming propensities respectively compared with those of α+β and α/β proteins. This may suggest that, with more complex architectures than α and β proteins, α+β and α/β proteins require larger propensities to distinguish from interacting α-helices and β-strands. Our finding of folding type-specific secondary structure propensities suggests that sequence space accessible to each folding type may have differing features. Differing sequence space features might be constrained by topological requirement for each of the folding types. Almost all strong β-sheet forming residues are hydrophobic in character regardless of folding types, thus suggesting the hydrophobicities of side chains as a key determinant of β-sheet structures. In contrast, conformational entropy of side chains is a major determinant of the helical propensities of amino acids, although other interactions such as hydrophobicities and charged interactions cannot be neglected. These results will be helpful to protein design, class-based secondary structure prediction, and protein folding. © 1998 John Wiley & Sons, Inc. Biopoly 45: 35–49, 1998     

Racemization and hydrolysis of tropic acid alkaloids in the presence of cyclodextrins

Because of the constantly increasing demand for optically pure drugs it is of great importance to elucidate factors affecting stereochemistry, in order to provide a stable formulation with a high chiral quality of the desired isomer. Therefore, the effects of cyclodextrins (CyDs) and their alkylated and hydroxyalkylated derivatives on racemization and hydrolysis of (?)-(S)-hyoscyamine and (?)-(S)-scopolamine were examined kinetically and spectroscopically (NMR). Direct methods, based on a chiral and achiral chromatographic phase system, were used to determine their degradation products and enantiomer composition during stability tests. All different CyDs, except α-CyD, retarded racemization and hydrolysis. The inclusion of the drug substances in CyDs inhibits the attack of hydroxyl ions and/or water molecules and thus retards the racemization and hydrolysis. The racemization of the tropic acid alkaloids is dependent on the pH and temperature. NMR studies were used to evidence the formation of a soluble 1:1 complex in aqueous solution. © 1993 Wiley-Liss, Inc.     

Association and dissociation properties of natural human interferon γ

The properties of natural human interferon γ (IFN-γ) molecules dissolved in protein-denaturing and non-denaturing solvents were examined by high-performance size-exclusion chromatography on a gel permeation column. IFN-γ and tritium-labeled IFN-γ molecules formed either dimers (>90.5%) with the molecular mass of 60 kDa or probably tetramers (<9.5%) with the molecular mass of approximately 100 kDa in non-denaturing solvents, and no monomer was detected. These oligomers were dissociated in protein-denaturing solvents such as 6 M guanidine hydrochloride, and IFN-γ existed as monomers. There is no effect on formation of the monomer based on the dissociation of oligomers by acid treatment at pH 4.0. The monomers in protein-denaturing solvents formed dimers by association when applied to a column equilibrated with a non-denaturing solvent of phosphate buffer, pH 7.0. In conclusion, natural human IFN-γ forms oligomers, particularly dimers, in non-denaturing solution, and this oligomer formation is a reversible reaction.     

A consensus prediction of the secondary structure for the 6-phospho-β-D-galactosidase superfamily

Two separate unrefined models for the secondary structure of two subfamilies of the 6-phospho-β-D -galactosidase superfamily were independently constructed by examining patterns of variation and conservation within homologous protein sequences, assigning surface, interior, parsing, and active site residues to positions in the alignment, and identifying periodicities in these. A consensus model for the secondary structure of the entire superfamily was then built. The prediction tests the limits of an unrefined prediction made using this approach in a large protein with substantial functional and sequence divergence within the family. The protein belongs to the (α–β class), with the core β strands aligned parallel. The supersecondary structural elements that are readily identified in this model is a parallel β sheet built by strands C, D, and E, with helices 2 and 3 connecting strands (C + D) and (D + E), respectively, and an analogous α–β unit (strand G and helix 7) toward the end of the sequence. The resemblance of the supersecondary model to the tertiary structure formed by 8-fold α–β barrel proteins is almost certainly not coincidental. © 1995 Wiley-Liss, Inc.     

Synthesis of phosphonic analogues of carnitine and gamma-amino-beta-hydroxybutyric acid

The involvement of carnitine and gamma-amino-beta-hydroxybutyric acid in the biology of mammalian cells, the physiology of the human body, and some important aspects of medicinal treatment has induced many research groups to develop their pharmacologically potent analogues. Among them are the very important phosphonic analogues: phosphocarnitine and gamma-amino-beta-hydroxypropylphosphonic acid. This mini-review describes the various methodologies used for the synthesis of these compounds.     

Structure,Function, and Regulation of the Three β-Adrenergic Receptors

Three β-adrenergic receptor subtypes are now known to be functionally expressed in mammals. All three belong to the R₇G family of receptors coupled to G-proteins, and characterized by an extracellular glycosylated N-terminal and an intracellular C-terminal region and seven transmembrane domains, linked by three exta- and three intracellular loops. The catecholamine ligand binding domain, studied using affinity-labeling and site-directed mutagenesis, is a pocket lined by residues belonging to the transmembrane domains. The region responsible for the interaction with the Gs protein which, when activated, stimulates adenylyl cyclase, is composed of residues belonging to the parts most proximal to the membrane of intracellular loop i₃ and the C-terminal region. The pharmacology of the three subtypes is quite distinct: in fact most of the potent β₁/β₂ antagonists (the well known β blockers) act as agonists on β₃. The subtype is resistant to short-term desensitization mediated by phosphorylation through PKA or βARK, in stark contrast to the β₁ or β₂ subtypes. Various compounds (dexamethasone, butyrate, insulin) up regulate β₁ or β₁ subtypes while down-regulating β₃ whose expression strictly correlates with differentiation of 3T3-F442A fibroblasts into adipocytes, thus confirming that the expression of the three subtypes may each be regulated independently to exert a specific physiologic role in different tissues or at different stages of development.     

Predicted topology of the N-terminal domain of the hydrophilic subunit of the mannose transporter of Escherichia coli

A folding topology for the homodimeric N-terminal domain (IIA, 2 × 14 kDa) of the hydrophilic subunit (IIAB^man) of the mannose transporter of E. coli is proposed. The prediction is based on (i) tertiary structure prediction methods, and (ii) functional properties of site-directed mutants in correlation with NMR-derived α/β secondary structure data. The 3D structure profile suggested that the overall fold of IIA is similar to that of the unrelated protein, flavodoxin, which is an open-stranded parallel β-sheet with a strand order of 5 4 3 1 2. The 3D model of IIA, constructed using the known atomic structure of flavodoxin, is consistent with the results from site-directed mutagenesis. Recently NMR results confirmed the open parallel β-sheet with a strand order of 4 3 12 (residues 1-120) of our model whereas β-strand 5 (residues 127–130) was shown to be antiparallel to β-strand 4. The correctly predicted fold includes 90% of the monomeric subunit sequence and contains all functional sites of the IIA domain.     

Immunostimulatory activities of monoglycosylated α-d-pyranosylceramides

We compared the immunostimulatory effects of chemically synthesized α-galactosylceramides (α-GalCers), α-glucosylceramides (α-GluCers), 6″-monoglycosylated α-GalCer and 6″- or 4″-monoglycosylated α-GluCer and made the following observations: (1) the length of the fatty acid side chain in the ceramide portions greatly affects the immunostimulatory effects of α-GalCers and α-GluCers; (2) the configuration of the 4″-hydroxyl group of the inner pyranose moiety plays an important role in the immunostimulatory effects of monoglycosylated α- -pyranosylceramides; (3) the free 4″-hydroxyl group of the inner pyranose of monoglycosylated α- -pyranosylceramides plays a more important role in their immunostimulatory effects than the free 6″-hydroxyl group.