Teicoplanin, a member of the "last chance" antibiotic family has a similar structure and the same mechanism of action as parent drug vancomycin, which is proved to be an effective binder of Cu(II) ions. However, the potentiometric and spectroscopic studies (UV-visible, CD, NMR) have shown that the modification of the N-terminal structure of the peptide backbone in teicoplanin affects considerably the binding ability towards Cu(II) ions. While vancomycin forms almost instantly the stable 3N complex species involving the N-terminal and two amide nitrogen donors, in case of teicoplanin only two nitrogen donors derived from the N-terminal amino group and adjacent peptide bond are coordinated to Cu(II) ion within the whole pH range studied. The major factor influencing the binding mode is most likely the structure of the N-terminus of the peptide unit in the antibiotic ligand.

The solution structure of kanamycin A interacting with a ribosomal A‐site fragment was solved by transferred‐NOE techniques and found to agree with the structure of the complex observed in the crystal. Despite the fast exchange conditions found for the interaction, the bound form was identified by NOESY spectroscopy. At 600 MHz, NOE effects are only observed for the RNA‐associated antibiotic. Dissociation constants were measured by NMR spectroscopy for two sites of interaction ( K d1 =150±40 μ M ; K d2 =360±50 μ M ). Furthermore, the effects of the Cu II ion on the antibiotic, on the RNA fragment that mimics the bacterial ribosomal A site, and on the complex formed between these two entities were analyzed. The study led to the proposal of a model that localizes the copper ion within the kanamycin–RNA complex.

Cyclosporin A (CsA) is a cyclic naturally occurring peptide used to prevent graft rejection in organ transplantations. Its immunosuppressive activity is due to the formation of a complex with cyclophilin A (Cyp), in which the cis 9MeLeu-10MeLeu amide bond of CsA assumes a trans conformation. The mechanism of the conformational inversion has not been delineated, but it has been postulated that metal ions binding induces a conformational change that enables CsA to bind Cyp. In this work, we solved the structures of CsA in sodium dodecyl sulfate (SDS) micelles (which enhance its solubility and mimic the hydrophobic environment clinically used for drug delivery) and its complex with Dy(III) ion, whose coordination chemistry is frequently used to reproduce the effect of Ca(II). The paramagnetic properties of Dy(III) allowed us to build up a structure using proton relaxation enhancements, which remains stable in a MD simulation in the micelle environment.

Tibolone is a synthetic molecule used extensively for the management of menopausal symptoms, with the proposed additional advantage of enhanced mood and libido. Tibolone, after oral administration, is rapidly converted into 3 major metabolites: 3alpha-hydroxytibolone and 3beta-hydroxytibolone, which have estrogenic effects, and the Delta(4)-isomer, which has progestogenic and androgenic effects. The tissue-selective effects of tibolone are the result of metabolism, of enzyme regulation, and of receptor activation which vary in different tissues. Tibolone seems to be effective on estrogen-withdrawal symptoms such as hot flushes, sweating, insomnia, headache, and vaginal dryness, with results generally comparable to the effects exerted by estrogen-based treatments, and the additional property of a progestogenic activity on the endometrium. As well as relieving vasomotor symptoms, tibolone has positive effects on sexual well-being and mood, and improves dyspareunia and libido. These effects may depend on both estrogenic and androgenic actions exerted at the genital level and in the central nervous system, and on a reduction of sex-hormone-binding globulin and an increase of free testosterone, without affecting Delta-5 androgens levels. Based on the evidence available, tibolone is a valuable treatment option to relieve menopausal complaints, especially in women suffering persistent fatigue, blunted motivation, and loss of sexual desire despite an adequate estrogen replacement.

The increased use of hormonal therapies has led to the study of the properties of different progestin molecules and their effects on the central nervous system. The central and peripheral levels of neurosteroid allopregnanolone and the opioid peptide β-endorphin (β-END) are regulated by estrogens. The aim of the present study was to investigate the effects of a 2-week oral treatment with micronized progesterone or medroxyprogesterone acetate (MPA) alone or in addition to estradiol valerate (E2V) on central and peripheral allopregnanolone and β-END levels in ovariectomized (OVX) female rats. Thirteen groups of Wistar OVX rats received one of the following treatments: oral progesterone (2, 4 or 8 mg/kg/day); oral MPA (0.05, 0.1 or 0.2 mg/kg/day); E2V (0.05 mg/kg/day); E2V + progesterone (0.05 mg/kg/day + 2, 4 or 8 mg/kg/day), or E2V + MPA (0.05 mg/kg/day + 0.05, 0.1 or 0.2 mg/kg/day) for 14 days. One group of fertile and one group of OVX rats were used as controls. The concentration of allopregnanolone was assessed in the frontal and parietal lobes, hypothalamus, hippocampus, anterior pituitary, adrenals and serum, while the β-END content was assessed in the frontal and parietal lobes, hypothalamus, hippocampus, anterior and neurointermediate pituitary, and plasma. E2V administration reverted the ovariectomy-induced reduction in allopregnanolone and β-END. Progesterone and MPA increased allopregnanolone levels in all tissues except in the adrenal gland. The combined administration of progesterone or MPA and E2V determined a further increase in allopregnanolone levels with respect to E2V alone except in the adrenal gland and hippocampus only after MPA treatment. Progesterone did not affect β-END levels in the frontal and parietal lobes, hippocampus and anterior pituitary, while it caused an increase plasma, hypothalamic and neurointermediate pituitary β-END levels. MPA only affected β-END levels in the hippocampus and in the neurointermediate lobe. The combined administration of progesterone or MPA and E2V did not alter the effect of estradiol or it determined a further dose-dependent increase in β-END levels. In conclusion, this study demonstrates that progesterone and MPA have a similar but not identical effect on central and peripheral allopregnanolone and β-END levels. Their association with an estrogenic compound does not interfere with the positive effects produced by estrogen on allopregnanolone and β-END brain content.

OBJECTIVE:The aging process is associated with a decline in the circulating Delta5-androgen dehydroepiandrosterone (DHEA) and its sulfate ester, dehydroepiandrosterone sulfate (DHEAS). The present study aimed to evaluate the effects of a long-term (12 months) oral DHEA administration (25 mg/day) on adrenal function, before and after 3, 6 and 12 months of treatment. METHOD:Postmenopausal women belonging to two age groups, 50-55 years (n = 10) and 60-65 years (n = 10), were studied. Adrenal function was assessed in basal conditions, after suppression with dexamethasone (DXM) and following a stimulation test with adrenocorticotropic hormone (ACTH) (10 microg bolus). Serum levels of DHEA, DHEAS, androstenedione (Delta4-A), allopregnanolone, 17-hydroxyprogesterone (17-OHP) and cortisol were measured and the effects of DHEA supplementation on specific adrenal enzymatic pathways were evaluated by calculating precursor/product ratios (17-OHP/cortisol, 17-OHP/Delta4-A, DHEA/Delta4-A and DHEA/DHEAS). RESULTS:DHEA supplementation annulled the age-related differences in DHEA and DHEAS levels and induced a marked increase in all steroids, except for cortisol, after 3-6 months of treatment. Serum cortisol levels decreased from the 3rd month, both in younger and older subjects. DHEA supplementation did not affect DXM-induced suppression of adrenal steroidogenesis. During the treatment period all adrenal androgens and progestins showed a significant increase in their response to ACTH, while the cortisol response decreased significantly. The results suggest a significant DHEA-induced change in adrenal enzymatic activities, as also evidenced by the change in precursor/product ratios during therapy. CONCLUSION:Chronic DHEA administration is capable of modifying circulating levels of androgens and progestins in both early and late postmenopausal women by modulating the age-related changes in adrenal function.

Cyclosporin A (CsA) is an important drug used to prevent graft rejection in organ transplantations. Its immunosuppressive activity is related to the inhibition of T-cell activation through binding with the proteins Cyclophilin (Cyp) and, subsequently, Calcineurin (CN). In the complex with its target (Cyp), CsA adopts a conformation with all trans peptide bonds and this feature is very important for its pharmacological action. Unfortunately, CsA can cause several side effects, and it can favor the excretion of calcium and magnesium. To evaluate the possible role of conformational effects induced by these two metal ions in the action mechanism of CsA, its complexes with Mg(II) and Ce(III) (the latter as a paramagnetic probe for calcium) have been examined by two-dimensional NMR and relaxation rate analysis. The conformations of the two complexes and of the free form have been determined by restrained molecular dynamics calculations based on the experimentally obtained metal-proton and interproton distances. The findings here ratify the formation of 1:1 complexes of CsA with both Mg(II) and Ce(III), with metal coordination taking place on carbonyl oxygens and substantially altering the peptide structure with respect to the free form, although the residues involved and the resulting conformational changes, including cis-trans conversion of peptide bonds, are different for the two metals.

Ferulic acid (Fer), a natural anti-oxidant and chemo-protector, is able to suppress experimental carcinogenesis in the forestomach, lungs, skin, tongue and colon. Several Fer derivatives have been suggested as promising candidates for cancer prevention, being the biological activity related also to the capacity of partitioning between aqueous and lipid phases. In the present work, pulsed field gradient (PFG) NMR diffusion measurement and NMR relaxation rates have been adopted for investigating the interaction of three Fer derivatives (Fer-C11, Fer-C12 and Fer-C13) with human erythrocytes. Binding to the erythrocyte membrane has been shown for all derivatives, which displayed a similar interaction mode such that the aromatic moiety and the terminal part of the alkyl chain were the most affected. Quantitative analysis of the diffusion coefficients was used to show that Fer-C12 and Fer-C13 display higher affinity for the cell membrane when compared with Fer-C11. These findings agree with the higher anti-oxidant activity of the two derivatives.

The central nervous system is one of the main target tissues for sex steroid hormones, which act on both through genomic mechanisms, modulating synthesis, release, and metabolism of many neuropeptides and neurotransmitters, and through non-genomic mechanisms, influencing electrical excitability, synaptic function, morphological features, and neuron-glia interactions. During the climacteric period, sex steroid deficiency causes many neuroendocrine changes. At the hypothalamic level, estrogen withdrawal gives rise to vasomotor symptoms, to eating behavior disorders, and altered blood pressure control. On the other hand, at the limbic level, the changes in serotoninergic, noradrenergic, and opioidergic tones contribute to the modifications in mood, behavior, and nociception. Hormone replacement therapy (HRT) positively affects climateric depression throughout a direct effect on neural activity and on the modulation of adrenergic and serotoninergic tones and may modulate the decrease in cognitive efficiency observed in climaterium. The identification of the brain as a de novo source of neurosteroids, suggests that the modifications in mood and cognitive performances occurring in postmenopausal women my also be related to a change in the levels of neurosteroids. These findings open new perspectives in the study of the effects of sex steroids on the central nervous system and on the possible use of alternative and/or auxiliary HRT.

The interaction between the single hexarepeat unit of chicken prion protein [ChPrP(54-59)] and Cu(II) was investigated by NMR, finding different coordination modes for the trans/trans and cis/trans isomers.

Although the contribution of androgens to female physical and mental well-being is still a matter of debate, recent studies support their therapeutic use, mainly to treat sexual dysfunction related to menopause.

The synthetic peptide encompassing residues 106-126 (PrP106-126, KTNMKHMAGAAAAGAVVGGLG) of the human prion protein was considered for its binding properties toward copper(II), manganese(II) and zinc(II) at pH 5.7. 1H and 13C 1D spectra, 1H spin-lattice relaxation rates, and 1H-15N and 1H-13C HSQC 2D experiments were obtained in the absence and in the presence of metal ions. While Zn(II) was found to yield negligible effects upon any NMR parameter, metal-peptide association was demonstrated by the paramagnetic effects of Cu(II) and Mn(II) upon 1D and 2D spectra. Delineation of structures of metal complexes was sought by interpreting the paramagnetic effect on 1H spin-lattice relaxation rates. Exchange of peptide molecules from the metal coordination sphere was shown to provide sizable contribution to the observed relaxation rates. Such contribution was calculated in the case of Cu(II); whereas the faster paramagnetic rates of peptide molecules bound to Mn(II) were determining spin-lattice relaxation rates almost exclusively dominated by exchange. Proton-metal distances were therefore evaluated in the case of the Cu(II) complex only and used as restraints in molecular dynamics calculations where from the structure of the complex was obtained. The peptide was shown to bind copper through the imidazole nitrogen and the ionized amide nitrogen of His-111 and the amino-terminal group with the terminal carboxyl stabilizing the coordination sphere through ionic interactions. The data were interpreted as to demonstrate that the hydrophobic C-terminal region was not affecting the copper-binding properties of the peptide and that this hydrophobic tail is left free to interact with other target molecules. As for the complex with Mn(II), qualitative information was obtained on carbonyl oxygens of Gly-124 and Leu-125, beyond the terminal Gly-126 carboxyl, being at close distance from the metal ion, that also interacts, most likely, through a hydrogen bond of metal-bound water, with the imidazole ring of His-111.

Allopregnanolone is the best characterized among neurosteroids, and its role in the control of neuroendocrine axes has attracted increasing interest recently. However, there is no available information about circulating levels of allopregnanolone during infancy, childhood and puberty. We studied two groups of children: 1) those aged between 0 and 2 y (n = 72), and 2) those aged between 6 and 18 y, at different Tanner's stages (n = 82). In each of these patients, serum allopregnanolone, progesterone, cortisol, and dehydroepiandrosterone levels were evaluated after informed consent; allopregnanolone was measured by RIA after acid extraction on cartridge. There was no significant variation of serum allopregnanolone levels either in male and female children during the first 2 y of life. Furthermore, although serum dehydroepiandrosterone levels showed a significant decrease, inversely correlated with age of the children (p < 0.01), serum cortisol and progesterone levels showed a significant age-related increase during the first 2 y of life. Cortisol and allopregnanolone levels were positively correlated (p < 0.01). During puberty, we observed a progressive increase in serum allopregnanolone levels in both boys and in girls, which were higher at Tanner's stage IV-V (0.7 ± 0.01 nM; mean ± SEM) than at stages I-II (0.32 ± 0.02 nM; p < 0.01); mean levels were significantly higher at puberty than in the first 2 y of life (p < 0.01). Furthermore, during puberty, serum progesterone and dehydroepiandrosterone levels also increased progressively with age in both boys and girls. Allopregnanolone and dehydroepiandrosterone levels were positively correlated throughout puberty. The present results indicate that serum allopregnanolone levels do not change during the first 2 y of life but increase during pubertal development, suggesting that this steroid may be involved in the adaptive neuroendocrine mechanisms related to puberty.

Age at diagnosis and karyotype phenotype correlations in 139 subjects with Turner syndrome were analyzed on the basis of data in the Human Cytogenetic Registry of North-Est Italy. In stressing the importance of early diagnosis, cytogenetic examination is recommended in all prepuberal girls with heigh < the 3-degrees centile.