The role of G protein-coupled estrogen receptor 1 on neurological disorders.

G protein-coupled estrogen receptor 1 (GPER) is a membrane-associated estrogen receptor (ER) associated with rapid estrogen-mediated effects. Over recent years GPER emerged has a potential therapeutic target to induce neuroprotection, avoiding the side effects elicited by the activation of classical ERs. The putative neuroprotection triggered by GPER selective activation was demonstrated in mood disorders, Alzheimer's disease or Parkinson's disease of male and female in vivo rodent models. In others, like ischemic stroke, the results are contradictory and currently there is no consensus on the role played by this receptor. However, it seems clear that sex is a biological variable that may impact the results. The major objective of this review is to provide an overview about the physiological effects of GPER in the brain and its putative contribution in neurodegenerative disorders, discussing the data about the signaling pathways involved, as well as, the diverse effects observed.

Ventral hernia mesh tack causes liver hemorrhage.

INTRODUCTION: The laparoscopic approach is an increasingly popular option for ventral hernia repair. In the wake of this new technology, unexpected complications have been reported. CASE PRESENTATION: We present the case of a patient who developed a liver laceration and hemorrhage after a mesh tacking device partially dislodged subsequent to ventral hernia repair. The patient underwent exploratory laparotomy, liver hemostasis and removal of the offending tack. DISCUSSION: Our patient partially dislodged a mesh tacking device likely after violent coughing during a bout of pneumonia. The exposed blade caused a liver laceration and hemorrhage. Few other unexpected complications of the use of mesh tacking devices have been noted in the literature. Tackless hernia repair has also been described. CONCLUSION: Laparoscopic ventral hernia repair with tacks may have unexpected complications of which the surgeon should be aware and advise patients. Our patient developed a liver laceration and symptomatic hemorrhage after partially dislodging a hernia mesh tack. Further research into tackless hernia repair may be beneficial. A low long-term recurrence rate would demonstrate if tackless hernia repair is a viable option.

GDNF contributes to oestrogen-mediated protection of midbrain dopaminergic neurones.

Parkinson's disease (PD) is characterised by the preferential loss of dopaminergic neurones from the substantia nigra (SN) that leads to the hallmark motor disturbances. Animal and human studies suggest a beneficial effect of oestrogen to the nigrostriatal system, and the regulation of neurotrophic factor expression by oestrogens has been suggested as a possible mechanism contributing to that neuroprotective effect. The present study was designed to investigate whether the neuroprotection exerted by 17ß-oestradiol on nigrostriatal dopaminergic neurones is mediated through the regulation of glial cell line-derived neurotrophic factor (GDNF) expression. Using an in vivo rat model of PD, we were able to confirm the relevance of 17ß-oestradiol in defending dopaminergic neurones against 6-hydroxydopamine (6-OHDA) toxicity. 17ß-oestradiol, released by micro-osmotic pumps, implanted 10 days before intrastriatal 6-OHDA injection, prevented the loss of dopaminergic neurones induced by 6-OHDA. 17ß-oestradiol treatment also promoted an increase in GDNF protein levels both in the SN and striatum. To explore the relevance of GDNF increases to 17ß-oestradiol neuroprotection, we analysed, in SN neurone-glia cultures, the effect of GDNF antibody neutralisation and RNA interference-mediated GDNF knockdown. The results showed that both GDNF neutralisation and GDNF silencing abolished the dopaminergic protection provided by 17ß-oestradiol against 6-OHDA toxicity. Taken together, these results strongly identify GDNF as an important player in 17ß-oestradiol-mediated dopaminergic neuroprotection.

17beta-estradiol induces transthyretin expression in murine choroid plexus via an oestrogen receptor dependent pathway.

Oestrogen protects against AD by multiple mechanisms, including the enhancement of Abeta clearance. Transthyretin (TTR) is a homotetrameric protein mainly synthesized by the liver and choroid plexus (CP) of the brain that sequesters the amyloid beta (Abeta) peptide. In this study we examined the effects of 17beta-estradiol (E2) on TTR protein and mRNA levels, in primary cultures of rat CP epithelial cells (CPEC) by Western blot and Real Time PCR, respectively. Moreover, the localization of oestrogen receptors alpha (ERalpha) and beta (ERbeta) in response to E2 treatment was analysed by confocal microscopy in these cells. The expression of TTR, ERalpha and ERbeta was also compared in the CP of castrated female mice treated with E2 to vehicle-treated animals by Real Time PCR. TTR concentration in the CSF of all these animals was measured by radioimmunoassay. E2 treatment induced TTR transcription and increased TTR protein content in CPEC. Pre-treatment with ICI 182,780 (ICI) abrogated E2-induced TTR expression suggesting that, TTR is up-regulated via an ER-dependent pathway. Confocal microscopy demonstrated extranuclear ERalpha and ERbeta localization in untreated CPEC. Upon E2 treatment, translocation of ERalpha to the nucleus occurred, while ERbeta remained in the cytosol. These data was concurrent with the up-regulation of TTR expression detected in the CP of castrated female mice subjected to E2 treatment. Our results highlight the importance of E2 on the regulation of TTR, which may participate in the oestrogen-induced decrease in Abeta levels and deposition described in the literature.

5Alpha-dihydrotestosterone up-regulates transthyretin levels in mice and rat choroid plexus via an androgen receptor independent pathway.

Transthyretin (TTR) is a 55 kDa plasma homotetrameric protein mainly synthesized in the liver and choroid plexuses (CPs) of the brain that, functions as a carrier for thyroxin and retinol binding protein. It sequesters amyloid beta (Abeta) peptide, and TTR levels in the cerebrospinal fluid (CSF) appear to be inversely correlated with Alzheimer's disease (AD) onset and progression. Androgen deprivation increases plasma Abeta levels, which indicate that androgens may reduce the levels of soluble Abeta, the peptide widely implicated in the initiation of AD pathogenesis; however, the underlying mechanisms are still poorly understood. In this study we examined the effects of 5alpha-dihydrotestosterone (DHT) on TTR protein and mRNA levels, in primary cultures of rat CPs epithelial cells (CPEC) by Western blot, and real time PCR, respectively. Moreover, TTR concentrations were measured in the CSF of castrated wild-type, and transgenic mice expressing human TTR subjected to DHT treatment, by radioimmunoassay and ELISA, respectively. TTR mRNA expression was also compared in the CPs, of the animals from each experimental group by real time PCR. DHT treatment increased TTR protein levels in CPEC, and induced TTR transcription in these cells. The combination of flutamide with DHT in the treatment of CPEC did not abrogate DHT-induced TTR levels, suggesting that TTR is up-regulated via an androgen receptor independent pathway. In the CPs of both mice strains, DHT also increased TTR mRNA levels, but no significant differences in TTR protein levels were detected in the CSF of these animals. These findings open a wide range of possibilities for future studies on Abeta deposition and cognitive function, in response to androgen induction of TTR in animal models of AD.

Transthyretin is up-regulated by sex hormones in mice liver.

Misfolding and aggregation of mutated and wild-type transthyretin (TTR) can cause familial amyloid polyneuropathy (FAP) and senile systemic amyloidosis (SSA), respectively. In some populations, FAP onset seems to occur on average 2-11 years earlier in men than in women, and SSA appears to be a disease of elderly men. Most (95-100%) SSA patients described in the literature are men, suggesting that amyloid deposition in these patients may be sex hormone related. On the basis of gender-related differences in FAP onset, and on the almost exclusivity of SSA in elder men, we hypothesize that, sex hormones may increase TTR synthesis by the liver, and therefore, may contribute to amyloid deposition. In order to test this hypothesis, castrated female and male mice were implanted with alzet mini-osmotic pumps, delivering 17beta-estradiol (E2) or 5alpha-dihydrotestosterone (DHT), or vehicle only, for 1 week. Sham operated animals were also included in the experiment. After hormonal stimulation, mice were euthanized under anaesthesia, and liver and sera were collected. The expression of TTR in liver, and the levels of TTR in sera in response to E2 and DHT were analysed by Real Time PCR and radioimmunoassay, respectively. Data analysis showed that, both hormones induced TTR transcription, which was concurrent with a consistent increase in the circulating levels of the protein. Taken together, all these data provide an indication that sex hormone stimulation may constitute a risk factor for SSA.

Transthyretin interacts with metallothionein 2.

Transthyretin (TTR) is a 55 kDa homotetrameric protein known for the transport of thyroxine and the indirect transportation of retinol. Within the central nervous system, TTR is primary synthesized and secreted into the cerebral spinal fluid by the choroid plexus (CP), whereas most TTR in the systemic circulation is produced and secreted by the liver. TTR is involved in two types of amyloid disease, the senile systemic amyloidosis and the familial amyloidotic polyneuropathy. TTR has also been implicated in the sequestration of amyloid beta peptide (Abeta), preventing its deposition. To explore other biological roles for TTR, we searched for protein-protein interactions using the yeast two-hybrid system with the full-length human TTR cDNA as bait. We found a novel interaction between TTR and metallothionein 2 (MT2) in human liver. This interaction was confirmed by competition binding assays, co-immunoprecipitation, cross-linking, and Western blotting experiments. Binding studies using MT1 showed a saturable specific interaction with TTR with a Kd of 244.8 +/- 44.1 nM. Western blotting experiments revealed a TTR-MT1/2 protein complex present in rat CP and kidney tissue extracts. Immunofluorescence experiments, in CP primary cell cultures and in CP paraffin sections, showed co-localization of TTR and MT1/2 in the cytoplasm of epithelial CP cells and localization of MT1/2 in the endoplasmic reticulum. Moreover, dot blot immunoassays of rat CSF provided the first evidence, to our knowledge, of circulating metallothionein in CSF. Taken together, we suggest that TTR-MT1/2 complexes may be functionally significant not only in healthy conditions but also in Abeta deposition in Alzheimer disease, thereby providing a novel potential therapeutic target.

Evidence for population level declines in adult HIV prevalence in Kenya.

The HIV/AIDS epidemic in Kenya has been tracked through annual sentinel surveillance in antenatal clinics since 1990. The system started with 13 sites and now has over 35. Behaviours have been measured through national Demographic and Health Surveys in 1993, 1998, and 2003. The surveillance data indicate that prevalence has declined substantially starting in 1998 in five of the original 13 sites and starting in 2000 in another four sites. No decline is evident in the other five original sites although the 2004 estimate is the lowest recorded. Nationally, adult prevalence has declined from 10% in the late 1990s to under 7% today. Surveys indicate that both age at first sex and use of condoms are rising and that the percentage of adults with multiple partners is falling. It is clear that HIV prevalence is now declining in Kenya in a pattern similar to that seen in Uganda but seven or eight years later. Although the coverage of preventive interventions has expanded rapidly since 2000 this expansion was too late to account for the beginnings of the decline in prevalence. More work is needed to understand fully the causes of this decline, but it is encouraging to see Kenya join the small list of countries experiencing significant declines in HIV prevalence.

Reconciling nuclear microsatellite and mitochondrial marker estimates of population structure: breeding population structure of Chesapeake Bay striped bass (Morone saxatilis).

Comparative analyses of nuclear and organelle genetic markers may help delineate evolutionarily significant units or management units, although population differentiation estimates from multiple genomes can also conflict. Striped bass (Morone saxatilis) are long-lived, highly migratory anadromous fish recently recovered from a severe decline in population size. Previous studies with protein, nuclear DNA and mitochondrial DNA (mtDNA) markers produced discordant results, and it remains uncertain if the multiple tributaries within Chesapeake Bay constitute distinct management units. Here, 196 young-of-the-year (YOY) striped bass were sampled from Maryland's Choptank, Potomac and Nanticoke Rivers and the north end of Chesapeake Bay in 1999 and from Virginia's Mataponi and Rappahannock Rivers in 2001. A total of 10 microsatellite loci exhibited between two and 27 alleles per locus with observed heterozygosities between 0.255 and 0.893. The 10-locus estimate of R(ST) among the six tributaries was -0.0065 (95% confidence interval -0.0198 to 0.0018). All R(ST) and all but one theta estimates for pairs of populations were not significantly different from zero. Reanalysis of Chesapeake Bay striped bass mtDNA data from two previous studies estimated population differentiation between theta=-0.002 and 0.160, values generally similar to mtDNA population differentiation predicted from microsatellite R(ST) after adjusting for reduced effective population size and uniparental inheritance in organelle genomes. Based on mtDNA differentiation, breeding sex ratios or gene flow may have been slightly male biased in some years. The results reconcile conflicting past studies based on different types of genetic markers, supporting a single Chesapeake Bay management unit encompassing a panmictic striped bass breeding population.

Differential contribution of syntaxin 1 and SNAP-25 to secretion in noradrenergic and adrenergic chromaffin cells.

We used botulinum neurotoxins (BoNT) to examine whether differences in the secretory activity of noradrenergic and adrenergic chromaffin cells are related to differences in the exocytotic machinery of these two types of bovine adrenal medulla cells. Cleavage of syntaxin and SNAP-25 by BoNT/C1 decreased in a dose-dependent way the release of both noradrenaline and adrenaline, but noradrenaline release was more sensitive to BoNT/C1. Cleavage of SNAP-25 by BoNT/A also had a larger inhibitory effect on noradrenaline release than on adrenaline release. Neither BoNT/C1 nor BoNT/A affected the intracellular Ca2+ responses induced by K+-depolarisation, and the extent of the inhibition of K+-evoked catecholamine release by selective blockers of voltage-gated Ca2+ channels was not affected by BoNT/C1. Therefore, our data do not support the hypothesis of a regulatory effect of syntaxin or SNAP-25 on the activity of Ca2+ channels. The lower sensitivity of adrenaline release to BoNT was not due to a reduced ability of the toxins to enter or to cleave their protein targets in adrenergic cells, since immunoblot analysis showed the cleavage of a larger fraction of syntaxin 1A in adrenergic cells, as compared to the cleavage in noradrenergic cells. The immunoblot analysis also showed larger amounts of syntaxin 1A in noradrenergic chromaffin cells than in adrenergic cells. Thus, in spite of a greater cleavage of syntaxin 1A in adrenergic cells by BoNT/C1, adrenaline release was less sensitive to BoNT/C1, suggesting that the release process in noradrenergic cells might be more dependent on syntaxin 1A and SNAP-25, as compared to adrenergic cells.

Li influx and binding, and li/mg competition in bovine chromaffin cell suspensions as studied by li NMR and fluorescence spectroscopy.

Li(+) influx by bovine chromaffin cells, obtained from bovine adrenal medulla, was studied in intact cell suspensions using (7)Li NMR spectroscopy with the shift reagent [Tm(HDOTP)](4-). The influx rate constants, k(i), were determined in the absence and in the presence of two Na(+) membrane transport inhibitors. The values obtained indicate that both voltage sensitive Na(+) channels and (Na(+)/K(+))-ATPase play an important role in Li(+) uptake by these cells. (7)Li NMR T(1) and T(2) relaxation times for intracellular Li(+) in bovine chromaffin cells provided a T(1)/T(2) ratio of 305, showing that Li(+) is highly, immobilized due to strong binding to intracellular structures. Using fluorescence spectroscopy and the Mg(2+) fluorescent probe, furaptra, the free intracellular Mg(2+) concentration in the bovine chromaffin cells incubated with 15 mM LiCl was found to increase by about mM after the intracellular Li(+) concentration reached a steady state. Therefore, once inside the cell, Li(+) is able to displace Mg(2+) from its binding sites.

Competition between Li+ and Mg2+ in neuroblastoma SH-SY5Y cells: a fluorescence and 31P NMR study.

Because Mg2+ and Li+ ions have similar chemical properties, we have hypothesized that Li+/Mg2+ competition for Mg2+ binding sites is the molecular basis for the therapeutic action of lithium in manic-depressive illness. By fluorescence spectroscopy with furaptra-loaded cells, the free intracellular Mg2+ concentration within the intact neuroblastoma cells was found to increase from 0. 39 +/- 0.04 mM to 0.60 +/- 0.04 mM during a 40-min Li+ incubation in which the total intracellular Li+ concentration increased from 0 to 5.5 mM. Our fluorescence microscopy observations of Li+-free and Li+-loaded cells also indicate an increase in free Mg2+ concentration upon Li+ incubation. By 31P NMR, the free intracellular Mg2+ concentrations for Li+-free cells was 0.35 +/- 0. 03 mM and 0.80 +/- 0.04 mM for Li+-loaded cells (final total intracellular Li+ concentration of 16 mM). If a Li+/Mg2+ competition mechanism is present in neuroblastoma cells, an increase in the total intracellular Li+ concentration is expected to result in an increase in the free intracellular Mg2+ concentration, because Li+ displaces Mg2+ from its binding sites within the nerve cell. The fluorescence spectroscopy, fluorescence microscopy, and 31P NMR spectroscopy studies presented here have shown this to be the case.

Two types of omega-agatoxin IVA-sensitive Ca2+ channels are coupled to adrenaline and noradrenaline release in bovine adrenal chromaffin cells.

To clarify the role of P-type Ca2+ channels in catecholamine release from adrenal chromaffin cells we examined the concentration dependence of the effect of omega-agatoxin IVA on the release both of adrenaline and noradrenaline induced by a K(+)-evoked depolarization. omega-Agatoxin IVA caused a biphasic dose-dependent inhibition of secretion with a high-potency component (IC50 < 1 nM), responsible for 10-15% of catecholamine release evoked by 70 mM K+, and a low-potency component that accounted for about 40% of release, with IC50 values of 57 nM and 48 nM for noradrenaline and adrenaline release, respectively. The release of catecholamines from chromaffin cells was also inhibited dose dependently by omega-conotoxin MVIIC with IC50 values of 182 and 218 nM for noradrenaline and adrenaline release, respectively. The effects of 3 nM omega-agatoxin IVA and 3 microM omega-conotoxin MVIIC were additive, indicating that at the concentrations used the toxins were acting at independent sites, presumably, P- and Q-type Ca2+ channels. The blockade of Q-type channels inhibited the release of adrenaline (72 +/- 4.1%) significantly more than the release of noradrenaline (50 +/- 2.7%), suggesting a higher density or a closer coupling of these channels to exocytosis in adrenergic chromaffin cells. The blockade of P-type channels caused a greater inhibition of catecholamine secretion at low levels of K(+)-evoked depolarization and shorter times of stimulation than that observed at higher levels of stimulation. The contribution of Q-type channels to catecholamine secretion did not change significantly with the intensity of stimulation. The data show that two types of omega-agatoxin IVA-sensitive Ca2+ channels are coupled to catecholamine release in chromaffin cells, and that the contribution of P-type channels to secretion is larger at low levels of depolarization.

Differential stimulation of noradrenaline release by reversal of the Na+/Ca2+ exchanger and depolarization in chromaffin cells.

We compared the effectiveness of Ca2+ entering by Na+/Ca2+ exchange with that of Ca2+ entering by channels produced by membrane depolarization with K+ in inducing catecholamine release from bovine adrenal chromaffin cells. The Ca2+ influx through the Na+/Ca2+ exchanger was promoted by reversing the normal inward gradient of Na+ by preincubating the cells with ouabain to increase the intracellular Na+ and then removing Na+ from the external medium. In this way we were able to increase the cytosolic free Ca2+ concentration ([Ca2+]c) by Na+/Ca2+ exchange to 325 +/- 14 nM, which was similar to the rise in [Ca2+]c observed upon depolarization with 35 mM K+ of cells not treated with ouabain. After incubating the cells with ouabain, K+ depolarization raised the [Ca2+]c to 398 +/- 31 nM, and the recovery of [Ca2+]c to resting levels was significantly slower. Reversal of the Na+ gradient caused an approximately 6-fold increase in the release of noradrenaline or adrenaline, whereas K+ depolarization induced a 12-fold increase in noradrenaline release but only a 9-fold increase in adrenaline release. The ratio of noradrenaline to adrenaline release was 1.24 +/- 0.23 upon reversal of the Na+/Ca2+ exchange, whereas it was 1.83 +/- 0.19 for K+ depolarization. Reversal of the Na+/Ca2+ exchange appeared to be as efficient as membrane depolarization in inducing adrenaline release, in that the relation of [Ca2+]c to adrenaline release was the same in both cases. In contrast, we found that for the same average [Ca2+]c, the Ca2+ influx through voltage-gated channels was much more efficient than the Ca2+ entering through the Na+/Ca2+ exchanger in inducing noradrenaline release from chromaffin cells.(ABSTRACT TRUNCATED AT 250 WORDS)