Epitope mapping and neuroprotective properties of a human single chain FV antibody that binds an internal epitope of amyloid-beta 1-42.

Active and passive immunotherapy targeted at the amyloid-beta (Abeta) peptide has been proposed as therapeutic approach against Alzheimer's disease (AD), and efforts towards the generation and application of antibody-based reagents that are capable of preventing and clearing amyloid aggregates are currently under active investigation. Previously, we selected and characterized a new anti-Abeta1-42 phage-displayed scFv antibody, designated clone b4.4, using a non-immune human scFv antibody library and demonstrated that a peptide based on the sequence of the Ig heavy chain (VH) complementarity-determining region (HCDR3) of this antibody fragment bound to Abeta1-42)and had neuroprotective potential against Abeta1-42 mediated neurotoxicity in rat hippocampal cultured neurons. In the present study, using novel computational methods and in vitro experiments we demonstrated that b4.4 binds to the central region of Abeta1-42. We also demonstrated that this scFv antibody binds to Abeta-derived diffusible ligands (ADDLs) and neutralizes the toxicity of both fibrillar and oligomeric forms of Abeta1-42 tested in vitro in SH-SY5Y cell cultures.

Hypothalamic monoamine oxidase activity in ovariectomized rats after sexual behavior restoration.

The effect of estradiol benzoate, progesterone and a sequential treatment with both on the activity of the enzyme monoamine-oxidase (MAO) was assessed in mitochondria from hypothalami of ovariectomized rats. A differential effect on the subtypes A and B MAO was found according to the type of treatment. Estradiol benzoate administration decreased MAO activity, mainly that of MAO-A. Progesterone alone had no effect, and sequential treatment with estradiol benzoate plus progesterone restored sexual behavior and produced a significant increase of MAO-A activity, without changes in total MAO activity. Since MAO-A is an isoform of MAO that preferentially uses norepinephrine and serotonin as substrates and MAO-B acts on phenylethylamine and benzylamine as substrates, our findings suggest that the restoration of sexual behavior after the treatment with estradiol benzoate followed by progesterone may be associated with the differential effect exerted by the hormones on MAO subtypes, rather than to the simple decrease in hypothalamic monoamine concentrations as reported in the literature.

Progesterone-like relaxant effect of RU 486 in the rat myometrium.

The antihormone RU 486 is characterized by its antiprogesterone and antiglucocorticoid activities. In this work the likelihood of a non-genomic effect for this compound was assessed. Thus, RU 486 was compared with progesterone and the 5 beta-progestin pregnanolone, for its ability to modify the uterine contractility of the rat. An outstanding relaxant effect elicited by RU 486 was observed, slightly higher than that produced by progesterone but lower than pregnanolone. Moreover, calcium promoted contractions were antagonized by RU 486, in the same way as the endogenous steroids. The data suggest the capability of RU 486 to block the calcium channels. It is concluded that a non-genomic effect of RU 486 is produced before its journey into the cell for its genomic action.

External calcium dependence of the uterine contraction induced by prostaglandins E2 and F2 alpha and its antagonism with natural progestins.

Prostaglandins (PGs) E2 and F2 alpha are strong inducers of uterine contraction by promoting a Ca2+ increase into the cell through specific receptors coupled with the calcium channels. On the contrary, progesterone and 5 beta-reduced progestins promote smooth muscle relaxation by blocking the ion calcium influx. Thus, this study was designed to emphasize the importance of external calcium in the PGs-induced rat uterus contraction. Likewise, also studied was the antagonism and the interaction between PGs and progestins (progesterone and its 5 alpha and 5 beta-reduced derivatives) in the myometrium. Results showed that uterine contraction induced by PGs depends on external calcium, since verapamil or extracellular calcium depletion abolished the PGs effect. Regarding the PGs-progestins antagonism, it was observed that pregnanedione, pregnanolone and epipregnanolone were quite effective for counteracting of PGs-induced contraction. However, progesterone was effective in a middle range, whereas 5 alpha-reduced progestins (allopregnanedione and allopregnanolone) were almost ineffective. It has been concluded that the participation of PGs and progestins in the modulation of uterine contraction might be achieved through the control of calcium influx by opening (PGs) or blocking (progestins) receptor-operated calcium channels.

Chemical structure of corticosteroids and its relationship with their acute induction of lordosis in the female rat.

The intravenous injection of several corticosteroids in the spayed estrogen-primed rat resulted, 5 min later, in a remarkable induction of lordosis response for deoxycorticosterone and 11-deoxycortisol. A lower but important effect was induced with corticosterone and 17 alpha-hydroxyprogesterone. A chemical structure-biological effect analysis showed that C-21 hydroxylation (e.g., deoxycorticosterone, 11-deoxycortisol, and corticosterone) induces the behavioral effect. However, C-11 or C-17 hydroxylation alone promotes the lordosis response to a lesser degree (corticosterone and 17 alpha-hydroxyprogesterone, respectively). However, the effect of these latter compounds is significant. From the results, it is concluded that deoxycorticosterone and 11-deoxycortisol along with other steroids such as 20 alpha-hydroxyprogesterone are suitable candidates for synergizing the well-known estrogen-progesterone induction of lordotic behavior.

Non-genomic mechanism of action of delta-4 and 5-reduced androgens and progestins on the contractility of the isolated rat myometrium.

Effective concentrations50 of androgens, i.e. testosterone, androsterone, androstanediol, 5 beta-dihydrotestosterone and progestins: progesterone, pregnanolone, pregnanedione, epipregnanolone, allopregnanolone and allopregnanedione were assayed on the tonic contractions of the isolated rat myometrium induced by calcium in high-potassium calcium-free depolarizant solutions. Steroids showed their relaxant effect by fadding the sustained contraction induced by calcium in a depolarized state. Also, the addition of the calcium ionophores A-23187 and X-537A reversed the steroid relaxant effect by increasing sharply the tonic contraction. The possibility of steroid-induced relaxation through release of noradrenaline or histamine was discarded by blocking their specific receptors. From the results it is concluded that delta-4 and 5-reduced androgens and progestins produce relaxation by a myogenic mechanism acting on the smooth muscle cell, most likely by directly blocking the calcium channels they causing modulation of: the contraction-relaxation cycle.

Relaxant action of androgens, progestins and corticosteroids on the isolated ileum of the guinea pig.

The action of three different kinds of progesterone related compounds was tested on the spontaneous contractility of the isolated guinea pig ileum. The results showed that this tissue was very sensitive to the action of androgens, progestins and corticosteroids. Relaxation was observed as dose-dependent; however, effective doses 50 were different, being related to the chemical structure of the compound. 5 beta-progestins were the most potent steroids followed by 5-reduced androgens (5 alpha or 5 beta). 4-en compounds such as testosterone, progesterone, 17 alpha-OH-progesterone, and corticosteroids were the lower in potency. 5 alpha-pregnanedione and pregnanediols were practically ineffective. The high sensitiveness of the ileum smooth muscle makes this organ a target for steroids. Thus, steroidal influence on the intestine might be related with contractility changes in some physiological circumstances, e.g., pregnancy.

Modulatory action of 5-reduced androgens and progestins on the excitability of CNS and smooth muscle.

A hypothesis is proposed for the physiological role of the many 5 alpha and 5 beta-reduced metabolites of both testosterone and progesterone. The effects of 5-reduced steroids are observed mostly in excitable tissues. The outstanding effects are depression of neuronal activity, inhibition of neurotransmitters and smooth muscle relaxation. A clear chemical-structure-biological-activity relationship is observed. Thus, 5 beta-progestins are the most prominent, whereas 5 alpha-3-keto compounds are practically ineffective. An intermediate effect is observed with the delta 4 precursors and compounds with the configuration 3 alpha-hydroxy-5 alpha. Membranal stabilization and a Ca2+ antagonism are proposed as mechanisms of action. The main conclusion is that testosterone and progesterone are pre-hormones in the membranal effects of 5-reduced steroids. Likewise, these compounds participate in the cellular control of calcium influx, thereby modulating excitability in general.

Relaxant action of androgens, progestins and corticosteroids on the isolated ileum of the guinea pig.

The action of three different kinds of progesterone related compounds was tested on the spontaneous contractility of the isolated guinea pig ileum. The results showed that this tissue was very sensitive to the action of androgens, progestins and corticosteroids. Relaxation was observed as dose-dependent; however, effective doses 50 were different, being related to the chemical structure of the compound. 5 beta-progestins were the most potent steroids followed by 5-reduced androgens (5 alpha or 5 beta). 4-en compounds such as testosterone, progesterone, 17 alpha-OH-progesterone, and corticosteroids were the lower in potency. 5 alpha-pregnanedione and pregnanediols were practically ineffective. The high sensitiveness of the ileum smooth muscle makes this organ a target for steroids. Thus, steroidal influence on the intestine might be related with contractility changes in some physiological circumstances, e.g., pregnancy.

Microcomputer acquisition and processing of uterine contractions.

A microcomputer system was implemented for reliable, fast and accurate study of in vitro myometrial activity. By the analog-to-digital conversion, uterine contractions are captured, digitized, stored in magnetic disks and subsequently recalled for its mathematical analysis. The system allows to calculate area under the curve, dose-response curves and other parameters, including complex analysis of myometrial activity. Precision and saving time are the main advantages of the system, and it can be used to study any kind of biological waves. Because its low cost and simplicity, this system seems to be suitable for laboratories of physiology, pharmacology or biophysics.