Ana Martinho

Neuroprotective and neuroregenerative properties of metallothioneins

Metallothioneins (MTs) are low‐molecular weight cysteine‐ and metal‐rich proteins with unquestionable metal binding capacity, antioxidant and anti‐inflammatory properties, and a clear involvement in diverse physiological actions as inhibition of proapoptotic mechanisms, enhancement of cell survival, and tissue regeneration. Concurrent with this wide array of functions, MT‐1/2 have been implicated in neuroprotection and neuroregeneration. The zinc binding capacity and antioxidant properties of MTs may account for most of their physiological features in the brain. However, some receptor‐mediated actions of MT‐1/2 have also been reported recently, a subject to be fully elucidated. This review analyses and updates the current knowledge on the actions of MTs related to neuroprotection and neuroregeneration in an effort to distinguish receptor‐mediated actions of MTs from those arising from its zinc binding capacity and its antioxidant properties.

Human metallothioneins 2 and 3 differentially affect amyloid-beta binding by transthyretin.

Transthyretin (TTR), an amyloid‐beta (Aβ) scavenger protein, and metallothioneins 2 and 3 (MT2 and MT3), low molecular weight metal‐binding proteins, have recognized impacts in Aβ metabolism. Because TTR binds MT2, an ubiquitous isoform of the MTs, we investigated whether it also interacts with MT3, an isoform of the MTs predominantly expressed in the brain, and studied the role of MT2 and MT3 in human TTR–Aβ binding. The TTR–MT3 interaction was characterized by yeast two‐hybrid assays, saturation‐binding assays, co‐immunolocalization and co‐immunoprecipitation. The effect of MT2 and MT3 on TTR–Aβ binding was assessed by competition‐binding assays. The results obtained clearly demonstrate that TTR interacts with MT3 with a Kd of 373.7u2003±u200360.2u2003nm. Competition‐binding assays demonstrated that MT2 diminishes TTR–Aβ binding, whereas MT3 has the opposite effect. In addition to identifying a novel ligand for TTR that improves human TTR–Aβ binding, the present study highlights the need to clarify whether the effects of MT2 and MT3 in human TTR–Aβ binding observed inu2003vitro have a relevant impact on Aβ deposition in animal models of Alzheimer’s disease.

Stress and Glucocorticoids Increase Transthyretin Expression in Rat Choroid Plexus via Mineralocorticoid and Glucocorticoid Receptors

Transthyretin (TTR) is a carrier for thyroid hormones and retinol binding protein. Several mutated forms of TTR cause familial amyloidotic polyneuropathy, an inheritable lethal disease. On the other hand, wild-type TTR has a protective role against Alzheimer’s disease. Despite its overall importance in normal animal physiology and in disease, few studies have focused on its regulation. An in silico analysis of the rat TTR gene revealed a glucocorticoid responsive element in the 3′ region of the first intron. Thus, we hypothesised that TTR could be regulated by glucocorticoid hormones and investigated the regulation of TTR expression in response to hydrocortisone in a rat choroid plexus cell line (RCP) and in primary cultures of choroid plexus epithelial cells (CPEC). In addition, the effect of psychosocial stress on TTR expression was analysed in rat liver, choroid plexus (CP) and cerebrospinal fluid (CSF). In RCP and CPEC cultures hydrocortisone upregulated TTR expression, an effect suppressed by glucocorticoid receptor and mineralocorticoid receptor antagonists. Moreover, induction of psychosocial stress increased TTR expression in liver, CP and CSF of animals subjected to acute and chronic stress conditions. Overall, we conclude that stress upregulates TTR expression in CP.

Determination of ketamine and its major metabolite, norketamine, in urine and plasma samples using microextraction by packed sorbent and gas chromatography-tandem mass spectrometry.

Ketamine is a club drug widely abused for its hallucinogenic effects, being also used as a date-rape drug in recent years. We have developed an analytical method using gas chromatography-tandem mass spectrometry (GC-MS/MS) for the identification and quantification of ketamine and its major metabolite in urine and plasma. No derivatization step is needed to accomplish analysis. The compounds were extracted from 0.25mL of sample using microextraction by packed sorbent on mixed mode (M1) cartridges. Calibration curves were linear in the range of 10-250ng/mL for urine and 10-500ng/mL for plasma, with determination coefficients higher than 0.99. The limit of detection (LOD) was 5ng/mL for both compounds in both specimens. Recoveries ranged from 63 to 101%, while precision and accuracy were below 14% and 15%, respectively. These low limits of detection and the quite high recoveries obtained, in very low sample amounts, allow detecting small quantities of the compounds, making this procedure suitable for those laboratories performing routine analysis in the field of forensic toxicology. Compared with existing methods, the herein described procedure is fast, since no derivatization step is required, and cost effective for the quantification of ketamine and norketamine in biological specimens by gas chromatography.

Analysis of Salvinorin A in urine using microextraction in packed syringe and GC–MS/MS

BACKGROUNDnThe aim of this work was to develop and validate a method for the determination of Salvinorin A in human urine using microextraction by packed sorbent (MEPS) and GC-MS/MS.nnnRESULTSnThe technique uses a sample volume as low as 0.2 ml, and the analyte was extracted using a C18 sorbent. The method showed to be linear between 20 and 1000 ng/ml and presented a LOD of 5 ng/ml. Intra- and inter-day precision and accuracy were acceptable. Absolute recoveries ranged from 71 to 80%.nnnCONCLUSIONnGC-MS/MS with MEPS demonstrated to be a fast and simple procedure for the quantification of Salvinorin A in urine. This is the first time that GC-MS/MS with MEPS was used for the determination of this compound in biological fluids. Furthermore, the device could be reused for up to 80 extractions, which accounted for a lower cost of analysis.

Progesterone Enhances Transthyretin Expression in the Rat Choroid Plexus In Vitro and In Vivo via Progesterone Receptor

Depletion of ovarian hormones 17β-estradiol (E2) and progesterone (P) after menopause may contribute to the decline in cognitive performance and increases the risk of Alzheimer’s disease (AD) in women, striking the importance of understanding the regulation of pivotal proteins involved in AD pathogenesis by ovarian hormones. Transthyretin (TTR) is now recognized as one of such proteins due to its ability to sequester and degrade amyloid β (Aβ) into less harmful peptides and preventing their aggregation. We have previously demonstrated that E2 enhances TTR expression. In this study, we investigate the effects of P on TTR expression in primary cultures of rat choroid plexus epithelial cells and in adult ovariectomized female rats. The results obtained demonstrate that, in vitro and in vivo, TTR is up-regulated by P. In addition, the mechanism underlying the response of TTR to P was investigated, and we provide evidence that this response is achieved through a progesterone receptor-mediated mechanism. Our results reinforce the importance of ovarian hormones on the regulation of TTR, which may reflect on the processing of Aβ peptides and consequently on AD onset and progression.

A unique natural selective kappa-opioid receptor agonist, salvinorin A, and its roles in human therapeutics

Until the mid-60s, only the Mazatecs, an indigenous group from Oaxaca, Mexico, used Salvia Divinorum (S. divinorum) due to its hallucinogen properties. Later it was found that the hallucinogen effects of this plant were caused by the presence of a neoclerodane diterpene Salvinorin A (salvinorin A), which is a highly selective agonist of kappa-opioid receptor (KOR) that cause more intense hallucinations than the common hallucinogens as lysergic acid, mushrooms, ecstasy and others. In fact, smoking of only 200-500xa0μg of S. divinorum leaves is enough to produce these effects thus making it the most potent natural occurring hallucinogen known. Due to its legal status in various countries, this compound has gained a worldwide popularity as a drug of abuse with an easy access through smartshops and internet. Furthermore, salvinorin A gathered an increased interest in the scientific community thanks to its unique structure and properties, and various studies demonstrated that salvinorin A has antinociceptive, antidepressant, in some circumstances pro-depressant and anti-addictive effects that have yielded potential new avenues for research underlying salvinorin A and its semi-synthetic analogs as therapeutic agents.

Glucocorticoids regulate metallothionein-1/2 expression in rat choroid plexus: effects on apoptosis

The choroid plexus (CP) participates in the synthesis, secretion and regulation of the cerebrospinal fluid, in the removal of its toxic compounds and in the regulation of the availability of essential metal ions to the brain. It expresses and secretes metallothioneins 1/2 (MT-1/2) which are key components in the maintenance of the central nervous system metal homeostasis and have anti-apoptotic properties, thereby protecting the brain. Glucocorticoids regulate MT-1/2 expression in several brain regions, but within the choroid plexuses (CPs) it remains unknown. Glucocorticoid levels increase in response to stress with implications in apoptosis. Further, CP expresses glucocorticoid (GR) and mineralocorticoid receptors (MR) turning it into likely glucocorticoid responsive structure. Data prompted us to study the regulation of MT-1/2 expression in response to glucocorticoids in the rat CP, and to investigate its implications in apoptosis. MT-1/2 protein and mRNA expression analysis showed that hydrocortisone up-regulates MT-1/2 expression in rat choroid plexus (RCP) cell line and in primary cultures of choroid plexus epithelial cells (CPEC) cultures via GR and MR. Also, incubation of RCP cells with hydrocortisone significantly diminished apoptosis, an effect eliminated by the addition of a MT-1/2 antibody. Moreover, induction of psychosocial stress, with concomitant rise of corticosterone levels, increased MT-1/2 expression in liver and in CP of male and female rats, with an exception observed in CP from males subjected to acute stress in which down-regulation in MT-1/2 expression occurred. Altogether, the results obtained demonstrated that stress/glucocorticoids regulate MT-1/2 expression in rat CP, with implications on apoptosis.

A distal estrogen responsive element upstream the cap site of human transthyretin gene is an enhancer-like element upon ERα and/or ERβ transactivation.

Previous studies reported that 17 beta-estradiol (E2) is responsible for the up-regulation of transthyretin (TTR) expression via an estrogen receptor (ER)-dependent pathway in rat choroid plexus (CP) and liver. A computer-assisted homology search identified a putative estrogen-responsive element (ERE) in the 5 flanking region of the human TTR (hTTR) gene (ERETTR), with the sequence aAGTCAAAGTGACCa, between -3406 and -3392 bp. Luciferase reporter assays and electrophoretic mobility shift (EMSA) and supershift analysis were carried out to investigate if E2 regulates TTR transcription via this putative ERE. Luciferase reporter assays in COS-7 cells were carried out with a plasmid construction where the TTR fragment containing the putative ERETTR was cloned in pGL2-promoter vector (pGL2-P) (pGL2-P/TTR), co-transfected with estrogen receptor α (ERα) and/or estrogen receptor β (ERβ) expression vectors. These assays demonstrated that, upon incubation with E2, one or both ERs (α and/or β) transactivate the reporter gene. The pGL2-P/TTR showed a significant transactivation of up to 6.8-fold, by E2, when co-transfected with ERβ, and up to 4-fold with ERα. Specific binding of ER (α and/or β) to ERETTR was demonstrated by EMSA and supershift assays confirmed the binding to ERα and/or ERβ. Our findings further suggest a mechanism underlying the regulation of TTR expression through the identification of a novel ERE in the TTR gene, which functions as an E2-dependent enhancer-like element.

Simultaneous Quantification of Antidepressants and Metabolites in Urine and Plasma Samples by GC–MS for Therapeutic Drug Monitoring

The aim of this work was the optimization and method validation for antidepressants (fluoxetine, venlafaxine, amitriptyline, mianserin, trimipramine, nortriptyline, mirtazapine, sertraline, dothiepin, citalopram, and paroxetine) and their metabolites in urine and plasma samples using gas chromatography–mass spectrometry (GC–MS). The antidepressants were extracted with a Strata™ X solid-phase extraction (SPE) cartridge prior to analysis. The method presented linearity within the studied ranges in both samples with quantification limits varying from 1 to 15xa0ngxa0mL−1 and determination coefficients (R2) higher than 0.99 for all analytes in all runs. The limits of detection ranged from 1 to 5xa0ngxa0mL−1 for the compounds under study. The recovery for urine samples ranged from 40 to 89% and for plasma samples varied from 68 to 98%. Precision and accuracy analysis showed acceptable coefficients of variation and relative error, fulfilling the criteria normally accepted in bioanalytical method validation. The method developed proved to be suitable for screening of the studied drugs in urine and plasma samples, proving to be sensitive and presenting appropriate selectivity and sensitivity, allowing detection of small amounts of the analytes.Graphical Abstract