Ahmed Fadiel

Estrogen-induced hypothalamic synaptic plasticity and pituitary sensitization in the control of the estrogen-induced gonadotrophin surge

Proper gonadal function requires coordinated (feedback) interactions between the gonads, adenohypophysis, and brain: the gonads elaborate sex steroids (progestins, androgens, and estrogens) and proteins (inhibin-activin family) during gamete development. In both sexes, the brain-pituitary gonadotrophin-regulating interaction is coordinated by estradiol through its opposing actions on pituitary gonadotrophs (sensitization of the response to gonadotrophin-releasing hormone [GnRH]) versus hypothalamic neurons (inhibition of GnRH secretion). This dynamic tension between the gonadotrophs and the GnRH cells in the brain regulates the circulating gonadotrophins and is termed reciprocal/negative feedback. In females, reciprocal/negative feedback dominates ∼90% of the ovarian cycle. In a spectacular exception, the dynamic tension is broken during the surge of circulating estrogen that marks follicle and oocyte(s) maturation. The cause is an estradiol-induced disinhibition of the GnRH neurons that releases GnRH secretion to the highly sensitized pituitary gonadotrophs that in turn release the gonadotrophin surge (the estrogen-induced gonadotrophin surge [EIGS], also known as positive feedback). Studies during the past 4 decades have shown this disinhibition to result from estrogen-induced synaptic plasticity (EISP), including a reversible ∼ 50% loss in arcuate nucleus synapses. The disinhibited GnRH secretion occurs during maximal gonadotroph sensitization and results in the EIGS. Specific immunoneutralization of estradiol blocks the EISP and EIGS. The EISP is accompanied by increases in insulinlike growth factor 1, polysialylated neural cell adhesion molecule, and ezrin, 3 proteins that the authors believe are the links between estrogen-induced astroglial extension and the EISP that releases GnRH secretion at the moment of maximal sensitization of the pituitary gonadotrophs. The result is the paradoxical surge of gonadotrophins at the peak of ovarian estrogen secretion and the triggering of ovulation. This enhanced understanding of the mechanics of gonadotrophin control clarifies elements of the involved feedback loops and opens the way to a better understanding of the neurobiology of reproduction.

Low-dose estrogen therapy for prevention of osteoporosis: working our way back to monotherapy.

The risks of low bone mineral density, osteoporosis and fractures, are major concerns in postmenopausal women. Although postmenopausal hormone therapy is effective for reducing these risks, safety issues have been raised by the results of studies such as the Womens Health Initiative. Although there are scientifically valid reasons to be wary of the general applicability of the Womens Health Initiative findings, the study has underscored the continuing need for research into new forms of menopausal hormone therapy. Low-dose transdermal estrogen monotherapy can preserve bone density while relieving vasomotor symptoms. Transdermal administration may offer advantages, including lack of first-pass liver metabolism, which permits the use of lower doses and avoids a negative impact on the lipid profile. Moreover, a recently published 2-year study of ultra-low-dose transdermal estrogen monotherapy in an older population similar to that of the WHI reported significant increases in bone mineral density, accompanied by significant reductions in markers of bone turnover, with no increased risk of endometrial hyperplasia or other side effects. Additional studies are warranted to shed further light on the possible benefits of low-dose estrogen monotherapy for the prevention of bone loss in postmenopausal women.

Estrogens regulate posttranslational modification of neural cell adhesion molecule during the estrogen-induced gonadotropin surge.

Estrogen-induced synaptic plasticity (EISP) in the periventricular area (PVA) of the hypothalamus is necessary for the preovulatory gonadotropin surge. Because in situ enzymatic desialization of hypothalamic polysialylated (PSA) neural cell adhesion molecule (NCAM) blocked EISP, we examined the presence and amount of NCAM isotopes, PSA-NCAM, and sialylation enzymes in microdissected mouse hypothalamus tissues from proestrous afternoon [peak of estrogens and nadir of arcuate nucleus (AN) synapses] and metestrous morning (nadir of estrogens and highest AN synapses). Immunohistochemistry confirmed immunoreactive (ir) PSA-NCAM staining in the perineural spaces of the PVA. The extent of staining was cycle dependent, with more dense and complete profiles of individual neurons limned by the ir-PSA-NCAM staining on proestrus and less on metestrus. Western blots showed that high levels of ir-PSA-NCAM on proestrus are accompanied by diminished ir-NCAM-140 and -180 but not ir-NCAM-120 and the reverse on metestrus (P < 0.05). To evaluate the increase of sialylated NCAM at the expense of desialylated protein, expression of the responsible polysialyltransferase enzymes polysialyltransferase (ST8Sia IV) and sialyltransferase (ST8Sia II) mRNA levels were measured using RT-PCR. Both polysialyltransferase and sialyltransferase mRNA are more abundant on proestrus than metestrus (P < 0.05), indicating that these enzymes are regulated by estrogens. These results support estrogen-regulated formation and extrusion of hydrophilic PSA-NCAM into perineural spaces in the PVA as part of the mechanism of EISP.

Ab initio study of alkylation of guanine-cytosine base pair by sulfur and nitrogen mustards.

Abstract Quantum modeling of the N7(G) alkylation of guanine-cytosine (G-C) base pair by sulfur (HD) and nitrogen mustard (HN2) was performed by using the Density Functional Theory (DFT) BPW91/6–31G++DP procedure. The vibrational IR and Raman spectra are discussed with regard to the N7 position of guanine when electrophilic HD+ episulfonium and HN2+ aziridinium attack the G-C base pair. Thermodynamic and polarizability considerations are also presented. The computed electronic chemical potential and the electrophilicity of the studied species indicate that an electronic transfer is produced from the nucleophile (G-C) base pair to the electrophile HD+ episulfonium or HN2+ aziridinium during the alkylation process.

Protozoan parasite aquaporins

Protozoan parasites are a major threat to human health with millions of fatalities worldwide, especially in nonindustrialized countries. Currently, there is no cure for many of these parasitic diseases. Consequently, there is an imperative to find treatment targets and develop novel drugs based on the proteins encoded in the genomes of these parasites. Aquaporins, members of membrane proteins discovered and characterized within the past 20 years, are the mechanism through which water is transported through living membranes. The presence of aquaporins explains disease etiology related to water physiology and presents new pharmacogenomic targets. In this article, we review the literature on aquaporins found in Apicomplexan, Kinetoplastida and Microsporidia parasites as potential drug targets. Furthermore, by analyzing protein motion dynamics, we identify impediments that need to be surmounted for developing effective drugs targeting the aquaglyceroporin of Plasmodium falciparum, the causative agent of the most fatal form of human malaria.

Modern Pathology: Protein Mis-Folding and Mis-Processing in Complex Disease

Electrostatic and electrochemical properties of bio-molecules, such as proteins, are governed by energy parameters that are, in part dependent on its folding. Disruption of this process can lead to the development of complex, multisystem diseases whose presentation may be organ-dependent. Examples include cystic fibrosis, alpha-1 antitrypsin deficiency, and Alzheimer disease. In addition to explaining exotic pathologic syndromes, an understanding of protein folding mechanisms may facilitate the understanding of less complex diseases and allow the development of novel therapeutic approaches.

Regulation and activation of ezrin protein in endometriosis

BACKGROUND Ezrin protein and its activated form phospho-ezrin play a role in cell morphology, motility and adhesiveness. In this study, we hypothesized that these proteins play a role in the pathogenesis of endometriosis by promoting adhesion and invasion of endometrial stromal cells (ESCs) in ectopic sites. METHODS We compared the expression of ezrin and phospho-ezrin in normal endometrium from women without endometriosis with their expression in eutopic and ectopic endometrial tissues from women with endometriosis, using immunohistochemistry and western blot analysis. Paired eutopic and ectopic endometrial tissue samples from women with endometriosis (n = 13) and normal endometrium from women without endometriosis (n = 12) were collected. Invasive potential of ESCs from each of these samples was compared using Matrigel membrane invasion assay. RESULTS Eutopic and ectopic endometrial tissues from women with endometriosis have higher ezrin and phospho-ezrin levels as confirmed by immunohistochemistry and western blot analysis (P < 0.05). The Matrigel membrane invasion assay revealed that ectopic ESCs have more invasive characteristics, more protrusions and higher ezrin staining than normal ESCs (P < 0.05). CONCLUSIONS Ezrin can be a potential marker for endometrial cell invasion and may play a role in the pathogenesis of endometriosis.

Phenytoin Is an Estrogen Receptor α-Selective Modulator That Interacts With Helix 12

Rationale: Phenytoin (Dilantin®; DPH) is used to treat epilepsy but causes estrogen agonist–antagonist-like side effects. We investigated the interaction of phenytoin with estrogen receptors (ERs) α and β by computational molecular docking, ER competition binding, transcriptional assays, and biological actions, comparing outcomes with estradiol (E2), estrone (E1), and tamoxifen (TMX). Experimental: (1) The DPH docking to 3-dimensional crystal structures of the ERα ligand-binding domain (LBD) showed a high degree of structural complementarity (−57.15 calculated energy units, approximating kcal/mol) with the ligand-binding pocket, including a contact at leucine (L540) in helix 12. Estrogen receptor β showed slightly less favorable interactions (−54.27 kcal/mol), without contacting L450. Estradiol, E1, and TMX contact points with ERα and ERβ do not include L450. (2) Cellular actions: Incubation of cells transfected with ERα or ERβ and a luciferase promoter phenytoin was several orders weaker than E2 as an agonist through ERα and had no effect through ERβ. However, phenytoin at clinical concentrations (10−11 to 10−6 mol/L) powerfully antagonized action of E2 on ERα-expressing cells. Similarly, phenytoin at clinically effective concentrations marginally induced alkaline phosphatase by ERα- and ERβ-expressing endometrial cancer cells but at doses well below clinical effectiveness blocked E2-induced alkaline phosphatase. (3) ER competition: In Scatchard plots comparing phenytoin with 17β-estradiol against endometrial cancer cell cytosol E2-alone more effectively displaced labeled E2 than phenytoin, but phenytoin was approximately equimolar effective to E2 in inhibiting E2’s displacement of the radiolabel, further confirming that phenytoin is a strong E2 antagonist. Conclusions: At clinically effective concentrations, phenytoin is a strong ERα cell antagonist but a many-fold weaker agonist. Although it interacts with ERβ LBD residues, phenytoin has no effects on ERβ-only expressing cells. Docking studies indicate phenytoin interacts with the ERα LBD at the hinge of helix 12 and could thereby interfere with the entry of other ER ligands or with the mobility of helix 12, either of which actions could explain phenytoin’s antagonism of ER-mediated E2 actions. Our results suggest an explanation for the broad profile of phenytoin’s actions and raise possibilities for the use of phenytoin or congeners in the clinical management of ERα-dependent conditions.

Minimally invasive method for murine brain fixation

Complete brain fixation can be achieved with transthoracic cardiac infusion without thoracotomy. Light and electron microscopy tissue sections reveal preservation of cytoplasmic and nuclear structure at all magnification levels. Punched samples were obtained from the fixed tissue specimens in precisely localized areas for study using electron microscopy. This perfusion fixation technique provides both faster tissue harvesting capability and higher quality tissue preservation, without the artifacts of brain swelling and ventricular dilation observed in direct cardiac perfusion. Acute, discrete change in brain tissue can be studied.

Expression and activation of the membrane-cytoskeleton protein ezrin during the normal endometrial cycle

OBJECTIVE To examine total ezrin expression (ezrin and phospho-ezrin) through the normal endometrial cycle and to correlate ezrin activation and localization with cytologic changes. DESIGN Experimental laboratory study. SETTING University medical centers. PATIENT(S) Reproductive-age women. INTERVENTION(S) A total of 36 samples of normal early, mid-, and late proliferative- and secretory-phase endometrium were studied for immunoreactive total ezrin (ir-T-ezrin) and phospho-ezrin (ir-p-ezrin) expression by histology, immunohistochemistry, and Western blotting. MAIN OUTCOME MEASURE(S) Total ezrin and phospho-ezrin expressions through the normal endometrial cycle. RESULT(S) Throughout the cycle ir-T-ezrin is present in the epithelium. The intensity and localization of both ir-ezrin and ir-p-ezrin vary greatly throughout the cycle. The main findings include the following: lateral localization of ir-ezrin/ir-p-ezrin in association with membrane specializations; dense staining around secretory vacuoles (secretory phase); dense staining of the apical surfaces, including microvilli and pinopodes of epithelial cells, especially during the mid- to late secretory phases; and the presence of ezrin in the glandular secretions. Immunoreactive total ezrin and ir-p-ezrin were not expressed by stromal fibroblasts. CONCLUSION(S) Ezrin is a prominent protein in the cycling endometrium. The most striking findings were the gravitation of ir-ezrin/ir-p-ezrin to the periphery of secretory vacuoles, localization on apical surfaces of the luminal epithelium, dense ezrin staining in secretory-phase epithelial cell plumes, and the presence of ir-ezrin/ir-p-ezrin in secretory-phase luminal secretions. These findings may have functional implications, especially for implantation biology.